Modified exendins and exendin agonists

ABSTRACT

Novel modified exendins and exendin agonists having an exendin or exendin agonist linked to one or more polyethylene glycol polymers, for example, and related formulations and dosages and methods of administration thereof are provided. These modified exendins and exendin agonists, compositions and methods are useful in treating diabetes and conditions that would be benefited by lowering plasma glucose or delaying and/or slowing gastric emptying or inhibiting food intake.

RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S.provisional patent application Ser. No. 60/132,018, filed Apr. 30, 1999,which application is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel modified exendins and exendinagonists having an exendin or exendin agonist peptide linked to one ormore polyethylene glycol polymers (or other molecular weight increasingagents), and related products and methods that are useful, for example,in the treatment of diabetes, including Type 1 and 2 diabetes, in thetreatment of disorders which would be benefited by agents which modulateplasma glucose levels, and in the treatment of disorders which would bebenefited by the administration of agents useful in modulating glucagonor triglyceride levels, or the rate of gastric emptying or food intake,including obesity, eating disorders, and insulin-resistance syndrome.

BACKGROUND

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art to the presently claimedinvention, nor that any of the publications specifically or implicitlyreferenced are prior art to that invention.

The exendins are peptides that are found in the salivary secretions ofthe Gila monster and the Mexican Bearded Lizard, reptiles that areendogenous to Arizona and Northern Mexico. Exendin-3 [SEQ. ID. NO. 1] ispresent in the salivary secretions of Heloderma horridum (Mexican BeadedLizard), and exendin-4 [SEQ. ID. NO. 2] is present in the salivarysecretions of Heloderm suspectum (Gila monster)(Eng, J., et al., J.Biol. Chem., 265:20259-62, 1990; Eng, J., et al., J. Biol. Chem.,267:7402-05, 1992). The amino acid sequence of exendin-3 is shown inFIG. 1. The amino acid sequence of exendin-4 is shown in FIG. 2.Exendin-4 was first thought to be a (potentially toxic) component of thevenom. It now appears that exendin-4 is devoid of toxicity, and that itinstead is made in salivary glands in the Gila monster.

The exendins have some sequence similarity to several members of theglucagon-like peptide family, with the highest homology, 53%, being toGLP-1 [7-36]NH₂ [SEQ. ID. NO. 3] (Goke, et al., J. Biol. Chem.,268:19650-55, 1993). GLP-1 [7-36]NH₂, also sometimes referred to asproglucagon [78-107] or simply “GLP-1”, has an insulinotropic effect,stimulating insulin secretion from pancreatic beta-cells; GLP-1 has alsobeen reported to inhibit glucagon secretion from pancreatic alpha-cells(Ørsov, et al., Diabetes, 42:658-61, 1993; D'Alessio, et al., J. Clin.Invest., 97:133-38, 1996). GLP-1 has been reported to inhibit gastricemptying (Willms B, et al., J. Clin. Endocrinol. Metab. 81 (1): 327-32,1996; Wettergren A, et al., Dig. Dis. Sci. 38 (4): 665-73, 1993), andgastric acid secretion (Schjoldager B T, et al., Dig. Dis. Sci. 34 (5):703-8, 1989; O'Halloran D J, et al., J. Endocrinol. 126 (1): 169-73,1990; Wettergren A, et al., Dig. Dis. Sci. 38 (4): 665-73, 1993)). GLP-1[7-37], which has an additional glycine residue at its carboxy terminus,is reported to stimulate insulin secretion in humans (Ørsov, et al.,Diabetes, 42:658-61, 1993). Other reports relate to the inhibition ofglucagon secretion (Creutzfeldt W O C, et al., Glucagonostatic actionsand reduction of fasting hyperglycemia by exogenous glucagon-likepeptide I (7-36) amide in Type 1 diabetic patients, Diabetes Care 1996;19(6):580-6), and a purported role in appetite control (Turton M D, etal., A role for glucagon-like peptide-1 in the central regulation offeeding, Nature 1996 January; 379(6560):69-72). A transmembraneG-protein adenylate-cyclase-coupled receptor, said to be responsible atleast in part for the insulinotropic effect of GLP-1, has reportedlybeen cloned from a beta-cell line (Thorens, Proc. Natl. Acad. Sci. USA89:8641-45, 1992). GLP-1 has been the focus of significant investigationin recent years due to its reported action on the amplification ofstimulated insulin production (Byrne M M, Goke B. Lessons from humanstudies with glucagon-like peptide-1: Potential of the gut hormone forclinical use. In: Fehmann H C, Goke B. Insulinotropic Gut HormoneGlucagon-Like Peptide 1. Basel, Switzerland: Karger, 1997:219-33).

GLP-1 has also been reported to restore islet glucose sensitivity inaging rats, restoring their glucose tolerance to that of younger rats(Egan J M, et al., Diabetologia 1997 June; 40 (Suppl 1):A130). However,the short duration of biological action of GLP-1 in vivo is one featureof the peptide that has hampered its development as a therapeutic agent.Various methods have been tried to prolong the half-life of GLP-1 orGLP-1 (7-37), including attempts to alter their amino acid sequences andto deliver them using certain formulations (see, e.g., European PatentApplication, entitled “Prolonged Delivery of Peptides,” by Darley, etal., publication number 0 619 322 A2, regarding the inclusion ofpolyethylene glycol in formulations containing GLP-1 (7-37)).

Pharmacological studies have led to reports that exendin-4 can act atGLP-1 receptors in vitro on certain insulin-secreting cells, atdispersed acinar cells from guinea pig pancreas, and at parietal cellsfrom stomach; the peptide is also reported to stimulate somatostatinrelease and inhibit gastrin release in isolated stomachs (Goke, et al.,J. Biol. Chem. 268:19650-55, 1993; Schepp, et al., Eur. J. Pharmacol.,69:183-91, 1994; Eissele, et al., Life Sci., 55:629-34, 1994). Exendin-3and exendin-4 were reportedly found to stimulate cAMP production in, andamylase release from, pancreatic acinar cells (Malhotra, R., et al.,Regulatory Peptides, 41:149-56, 1992; Raufman, et al., J. Biol. Chem.267:21432-37, 1992; Singh, et al., Regul. Pept. 53:47-59, 1994).Exendin-4 has a significantly longer duration of action than GLP-1. Forexample, in one experiment, glucose lowering by exendin-4 in diabeticmice was reported to persist for several hours, and, depending on dose,for up to 24 hours (Eng, J. Prolonged effect of exendin-4 onhyperglycemia of db/db mice, Diabetes 1996 May; 45 (Suppl 2):152A(abstract 554)). Based on their insulinotropic activities, the use ofexendin-3 and exendin-4 for the treatment of diabetes mellitus and theprevention of hyperglycemia has been proposed (Eng, U.S. Pat. No.5,424,286).

The results of an investigation which showed that exendins are not thespecies homolog of mammalian GLP-1 was reported by Chen and Drucker whocloned the exendin gene from the Gila monster (J. Biol. Chem.272(7):4108-15 (1997)). The observation that the Gila monster also hasseparate genes for proglucagons (from which GLP-1 is processed), thatare more similar to mammalian proglucagon than exendin, indicated thatexendins are not merely species homologs of GLP-1.

Methods for regulating gastrointestinal motility using exendin agonistsare described in commonly owned U.S. patent application Ser. No.08/908,867, filed Aug. 8, 1997 entitled “Methods for RegulatingGastrointestinal Motility,” which application is a continuation-in-partof U.S. patent application Ser. No. 08/694,954, filed Aug. 8, 1996.

Methods for reducing food intake using exendin agonists are described incommonly owned U.S. patent application Ser. No. 09/003,869, filed Jan.7, 1998, entitled “Use of Exendin and Agonists Thereof for the Reductionof Food Intake,” which claims the benefit of U.S. ProvisionalApplication Nos. 60/034,905 filed Jan. 7, 1997, 60/055,404 filed Aug. 7,1997, 60/065,442 filed Nov. 14, 1997 and 60/066,029 filed Nov. 14, 1997.

Novel exendin agonist compounds are described in commonly owned PCTApplication Serial No. PCT/US98/16387 filed Aug. 6, 1998, entitled“Novel Exendin Agonist Compounds,” which claims the benefit of U.S.Patent Application Ser. No. 60/055,404, filed Aug. 8, 1997.

Other novel exendin agonists are described in commonly owned PCTApplication Serial No. PCT/US98/24210, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds,” which claims the benefit of U.S.Provisional Application No. 60/065,442 filed Nov. 14, 1997.

Still other novel exendin agonists are described in commonly owned PCTApplication Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds,” which claims the benefit of U.S.Provisional Application No. 60/066,029 filed Nov. 14, 1997.

Other recent advances in exendin related technology are described inU.S. Provisional Patent Application Ser. No. 60/075,122, filed Feb. 13,1998, entitled “Inotropic and Diuretic Effects of Exendin and GLP-1” andin U.S. Provisional Patent Application Ser. No. 60/116,380, filed Jan.14, 1998, entitled “Novel Exendin Agonist Formulations and Methods ofAdministration Thereof”.

Polyethylene glycol (PEG) modification of therapeutic peptides andproteins may yield both advantages and disadvantages. While PEGmodification may lead to improved circulation time, reduced antigenicityand immunogenicity, improved solubility, resistance to proteolysis,improved bioavailability, reduced toxicity, improved stability, andeasier formulation of peptides (See, Francis et al., InternationalJournal of Hematology, 68:1-18, 1998) problems with PEGylation in mostcases is substantial reduction in bioactivity. Id. In addition, mostmethods involve use of linkers that have several types of adverseeffects including immunogenicity, instability, toxicity, and reactivity.Id.

Modified exendins and exendin agonists and related formulations, dosageformulations, and methods that solve these problems and that are usefulin the delivery of therapeutically effective amounts of exendins andexendin agonists are described and claimed herein.

The contents of the above-identified articles, patents, and patentapplications, and all other documents mentioned or cited herein, arehereby incorporated by reference in their entirety. The inventorsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other documents mentioned or cited herein.

SUMMARY OF THE INVENTION

The present invention relates to novel modified exendins and exendinagonists having an exendin or exendin agonist linked to one or moremolecular weight increasing compounds, of which polyethylene glycolpolymers (or other molecular weight increasing agents), and relatedproducts and methods. Such products and methods that are useful for manyapplications, including, for example, in the treatment of diabetes,including Type 1 and 2 diabetes, gestational diabetes (see U.S. patentapplication Ser. No. 09/323,867, entitled, “Use of Exendins and AgonistsThereof For The Treatment of Gestational Diabetes Mellitus,” filed Jun.1, 1999), in the treatment of disorders which would be benefited byagents which modulate plasma glucose levels, in the treatment ofdisorders which would be benefited by the administration of agentsuseful in modulating the rate of gastric emptying or food intake,including obesity, eating disorders, and insulin-resistance syndrome,and to modulate triglyceride levels and to treat subjects suffering fromdyslipidemia (i.e., increased LDL cholesterol, increased VLDLcholesterol, and/or decreased HDL cholesterol)(see U.S. provisionalpatent application Ser. No. 60/175,365, entitled, “Use of Exendins andAgonists Thereof for Modulation of Triglyceride Levels and Treatment ofDyslipidemia,” filed Jan. 10, 2000). The methods are also useful forlowering plasma lipid levels, reducing cardiac risk, reducing theappetite, and reducing the weight of subjects. Still other embodimentsconcern methods for suppressing glucagon secretion (see U.S. provisionalpatent application Ser. No. 60/132,017, entitled, “Methods for GlucagonSuppression,” filed Apr. 30, 1999, which is commonly owned).Pharmaceutical compositions for use in the methods of the invention arealso disclosed.

The present invention is related to the surprising discovery thatexendin is cleared from the plasma almost entirely by renal filtration,and not primarily by proteolytic degradation, as occurs for many otherbiologically active peptides, for example, GLP-1. This surprisingdiscovery supports the determination that PEGylation or othermodification of exendin or exendin agonists to increase molecular size,will have pharmaceutical benefit.

Thus, the present invention provides a modified exendin or exendinagonist having an exendin or exendin agonist linked to one or morepolyethylene glycol polymers or other molecular weight increasingcompounds. A “molecular weight increasing compound” is one that can beconjugated to an exendin or exendin agonist and thereby increase themolecular weight of the resulting conjugate. Representative examples ofmolecular weight increasing compounds, in addition to PEG, are polyaminoacids (e.g., poly-lysine, poly-glutamic acid, and poly-aspartic acid;see Gombotz, et al. (1995), Bioconjugate Chem., vol. 6: 332-351; Hudecz,et al. (1992), Bioconjugate Chem., vol. 3, 49-57; Tsukada, et al.(1984), J. Natl. Cancer Inst., vol 73,: 721-729; Pratesi, et al. (1985),Br. J. Cancer, vol. 52: 841-848), particularly those of the Lconformation, pharmacologically inactive proteins (e.g., albumin; seeGombotz, et al. (1995) and the references cited therein), gelatin (seeGombotz, et al. (1995) and the references cited therein),succinyl-gelatin (see Gombotz, et al. (1995) and the references citedtherein), (hydroxypropyl)-methacrylamide (see Gombotz, et al. (1995) andthe references cited therein), a fatty acid, a olysaccaride, a lipidamino acid, and dextran.

In preferred embodiments, the modified exendin or exendin agonist has amolecular weight that is greater than the molecular weight of theexendin or exendin agonist (preferably about 10%, 50% or 90% greater),the modified exendin or exendin agonist has a negative charge that isgreater than the negative charge of the exendin or exendin agonist(preferably about 10%, 50% or 90% greater), the modified exendin orexendin agonist has a kidney clearance that is less than the kidneyclearance of the exendin or exendin agonist (preferably about 10%, 50%or 90% less), the modified exendin or exendin agonist has a half-lifethat is greater than the half-life of the exendin or exendin agonist(preferably about 10%, 50% or 90% greater), the modified exendin orexendin agonist has a immunogenicity/antigenicity that is less than theimmunogenicity/antigenicity of the exendin or exendin agonist, themodified exendin or exendin agonist has a solubility that is greaterthan the solubility of the exendin or exendin agonist (preferably about10%, 50% or 90% greater), the modified exendin or exendin agonist has aproteolysis rate that is less than the proteolysis rate of the exendinor exendin agonist (preferably about 10%, 50% or 90% less), the modifiedexendin or exendin agonist has a toxicity that is less than the toxicityof the exendin or exendin agonist, the modified exendin or exendinagonist has a stability that is greater than the stability of theexendin or exendin agonist, and/or the modified exendin or exendinagonist has a permeability/biological function that is greater or lessthan the permeability/biological function of the exendin or exendinagonist (preferably about 10%, 50% or 90% greater or less).

The exendin or exendin agonist may be linked to one, two or threepolyethylene glycol polymers or other molecular weight increasingagents. The polyethylene glycol polymers (or other molecular weightincreasing agents) may preferably have molecular weights between 500 and20,000. In a preferred embodiment, the modified exendin or exendinagonist is one of compounds 201-230, more preferably one of compounds209, 210 and 213, or one of compounds 201 and 202, or one of compounds216 and 217 (See Example 4 below).

The polyethylene glycol polymers (or other molecular weight increasingagents) are preferably linked to an amino, carboxyl, or thio group, andmay be linked by N or C termini of side chains of lysine, aspartic acid,glutamic acid, or cysteine, or alternatively, the polyethylene glycolpolymers or other molecular weight increasing agents may be linked withdiamine and dicarboxylic groups. The exendin or exendin agonist ispreferably linked to the polyethylene glycol polymers or other molecularweight increasing agents through an epsilon amino group on a lysineamino acid of the exendin or exendin agonist.

The present invention also features a method of making a modifiedexendin or exendin agonist. The method involves linking one or morepolyethylene glycol polymers or other molecular weight increasing agentsto an exendin or exendin agonist. In preferred embodiments, the linkingis performed by solid-phase synthesis.

The present invention also provides a method of treating a diseasebenefited by administration of an exendin or exendin agonist. The methodinvolves providing a modified exendin or exendin agonist of theinvention to a patient having such a disease and thereby treating thedisease. Exemplary diseases include postprandial dumping syndrome,postprandial hyperglycemia, impaired glucose tolerance, a condition ordisorder which can be alleviated by reducing food intake, obesity, aneating disorder, insulin-resistance syndrome, diabetes mellitus, and ahyperglycemic condition. In a preferred embodiment, the postprandialhyperglycemia is a consequence of Type 2 diabetes mellitus. In otherpreferred embodiments, the postprandial hyperglycemia is a consequenceof Type 1 diabetes mellitus or impaired glucose tolerance.

Also featured in the present invention is a pharmaceutical composition.The composition contains a modified exendin or exendin agonist and apharmaceutically acceptable carrier.

The invention also provides a kit. The kit contains a modified exendinor exendin agonist and instructions and/or packaging for use. The kitmay also include a document indicating that the kit, its components, orthe methods of using them, has received regulatory approval.

The present invention also provides a method of beneficially regulatinggastro-intestinal motility in a subject. The method involvesadministering to the subject a therapeutically effective amount of amodified exendin or exendin agonist of the present invention.

Also featured are methods of treatment for ingestion of a toxin. Themethods involve: (a) administering an amount of a modified exendin orexendin agonist of the present invention effective to prevent or reducethe passage of stomach contents to the intestines; and (b) aspiratingthe contents of the stomach.

The invention also provides methods for reducing the appetite or weight,or lowering plasma lipids, of a subject, as well as methods for treatinggestational diabetes. The invention also provides methods for reducingthe appetite or weight, or lowering plasma lipids, of a subject, as wellas methods for treating gestational diabetes. Additional methods includemodulating triglyceride levels, and treating subjects suffering fromdyslipidemia, as well as suppressing glucagon levels. These and othermethods of the invention involve administering to the subject atherapeutically effective amount of a modified exendin or exendinagonist of the present invention.

Modified exendins and exendin agonists are useful, for example, asinhibitors of gastric emptying for the treatment of, for example,diabetes mellitus, and obesity. Thus, the present invention is alsodirected to novel methods for reducing gastric motility and slowinggastric emptying. The methods involve the administration of a modifiedexendin or exendin agonist, for example one or more PEG polymers linkedto exendin-3 [SEQ ID NO. 1], exendin-4 [SEQ ID NO. 2], or othercompounds which effectively bind to the receptor at which exendins exerttheir action on gastric motility and gastric emptying. These methodswill be useful in the treatment of, for example, post-prandialhyperglycemia, a complication associated with type 1 (insulin dependent)and type 2 (non-insulin dependent) diabetes mellitus, as well asgestational diabetes, dyslipidemia, to modulate triglyceride levels, andto suppress glucagon secretion.

By “exendin agonist” is meant a compound which mimics the effects ofexendins, e.g., on gastric motility and gastric emptying (namely, acompound which effectively binds to the receptor at which exendins exerttheir action on gastric motility and gastric emptying, preferably ananalog or derivative of an exendin) or a compound, e.g., that mimics theeffects of exendin on the reduction of food intake by binding to thereceptor or receptors where exendin causes this effect. Preferredexendin agonist compounds include those described in United StatesPatent Application Ser. No. 90/003,869, entitled, “Use of Exendin AndAgonists Thereof For The Reduction of Food Intake”, filed Jan. 7, 1998,(and the priority applications thereto) which enjoys common ownershipwith the present application and which is incorporated by this referenceinto the present application as though fully set forth herein. Effectsof exendins or exendin agonists on reducing food intake can beidentified, evaluated, or screened for, using the methods describedherein, or other methods known in the art for determining exendineffects, e.g., on food intake or appetite.

In another aspect, a therapeutically effective amount of an amylinagonist is also administered to the subject. In a preferred aspect, theamylin agonist is an amylin or an amylin agonist analog such as^(25,28,29)Pro-human-amylin. The use of amylin agonists to treatpost-prandial hyperglycemia, as well as to beneficially regulategastrointestinal motility, is described in International Application No.PCT/US94/10225, published Mar. 16, 1995 which has been incorporated byreference herein.

In yet another aspect, a therapeutically effective amount of an insulinor insulin analog is also administered, separately or together with amodified exendin or exendin agonist, to the subject.

Preferably, the subject is a vertebrate, more preferably a mammal, andmost preferably a human. In preferred aspects, the modified exendin orexendin agonist of the invention is administered parenterally, morepreferably by injection. In a most preferred aspect, the injection is aperipheral injection. Preferably, about 1 μg-30 μg to about 5 mg of themodified exendin or exendin agonist of the invention is administered perday. More preferably, about 1-30 μg to about 2 mg, or about 1-30 μg toabout 1 mg of the modified exendin or exendin agonist of the inventionis administered per day. Most preferably, about 3 μg to about 500 μg ofthe modified exendin or exendin agonist of the invention is administeredper day.

Preferred exendins or exendin agonists for modification and use include:

exendin-4 (1-30) [SEQ ID NO 3: His Gly Glu Gly Thr Phe Thr Ser AspLeu Ser Lys Gln Met Glu Glu Glu Ala Val Arg LeuPhe Ile Glu Trp Leu Lys Asn Gly Gly]; exendin-4 (1-30) amide[SEQ ID NO 4: His Gly Glu Gly Thr Phe Thr Ser AspLeu Ser Lys Gln Met Glu Glu Glu Ala Val Arg LeuPhe Ile Glu Trp Leu Lys Asn Gly Gly-NH₂]; exendin-4 (1-28) amide[SEQ ID NO 5: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg LeuPhe Ile Glu Trp Leu Lys Asn-NH₂]; ¹⁴Leu, ²⁵Phe exendin-4 amide[SEQ ID NO 6: His Gly Glu Gly Thr Phe Thr Ser AspLeu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg LeuPhe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser SerGly Ala Pro Pro Pro Ser-NH₂]; ¹⁴Leu, ²⁵Phe exendin-4 (1-28) amide[SEQ ID NO 7: His Gly Glu Gly Thr Phe Thr Ser AspLeu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg LeuPhe Ile Glu Phe Leu Lys Asn-NH₂]; and¹⁴Leu, ²²Ala, ²⁵Phe exendin-4 (1-28) amide[SEQ ID NO 8: His Gly Glu Gly Thr Phe Thr Ser AspLeu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg LeuAla Ile Glu Phe Leu Lys Asn-NH₂].

In the methods of the present invention, the modified exendins orexendin agonists may be administered separately or together with one ormore other compounds and compositions that exhibit a long term orshort-term satiety action, including, but not limited to other compoundsand compositions that include an amylin agonist, cholecystokinin (CCK),or a leptin (ob protein). Suitable amylin agonists include, for example,[^(25,28,29)Pro-]-human amylin (also known as “pramlintide,” andpreviously referred to as “AC-137”) as described in “Amylin AgonistPeptides and Uses Therefor,” U.S. Pat. No. 5,686,511, issued Nov. 11,1997, and salmon calcitonin. The CCK used is preferably CCK octopeptide(CCK-8). Leptin is discussed in, for example, Pelleymounter, M. A., etal. Science 269:540-43 (1995); Halaas, J. L., et al. Science 269:543-46(1995); and Campfield, L. A., et al. Eur. J. Pharmac. 262:133-41 (1994).

The invention also provides compositions and methods for providingtherapeutically effective amounts of the modified exendins or exendinagonists of the invention in order to increase urine flow in anindividual, decrease the amount of potassium in the urine of anindividual, prevent or alleviate a condition or disorder associated withhypervolemia or toxic hypervolemia in an individual, induce rapiddiuresis, prepare an individual for a surgical procedure, increase renalplasma flow and glomerular filtration rates, or treat pre-eclampsia oreclampsia of pregnancy.

DEFINITIONS

In accordance with the present invention and as used herein, thefollowing terms are defined to have the following meanings, unlessexplicitly stated otherwise.

The term “amino acid” refers to natural amino acids, unnatural aminoacids, and amino acid analogs, all in their D and L stereoisomers iftheir structure allow such stereoisomeric forms. Natural amino acidsinclude alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid(Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine(Gly), histidine (His), isoleucine (Ile), leucine (Leu), Lysine (Lys),methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser),threonine (Thr), typtophan (Trp), tyrosine (Tyr) and valine (Val).Unnatural amino acids include, but are not limited toazetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyricacid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyricacid, 3-aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine,2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid,2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine,homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine,N-methylglycine, N-methylisoleucine, N-methylpentylglycine,N-methylvaline, naphthalanine, norvaline, norleucine, ornithine,pentylglycine, pipecolic acid and thioproline. Amino acid analogsinclude the natural and unnatural amino acids which are chemicallyblocked, reversibly or irreversibly, or modified on their N-terminalamino group or their side chain groups, as for example, methioninesulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine,S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteinesulfone.

The term “amino acid analog” refers to an amino acid wherein either theC-terminal carboxy group, the N-terminal amino group or side chainfunctional group has been chemically codified to another functionalgroup. For example, aspartic acid-(beta-methyl ester) is an amino acidanalog of aspartic acid; N-ethylglycine is an amino acid analog ofglycine; or alanine carboxamide is an amino acid analog of alanine.

The term “amino acid residue” refers to radicals having the structure:(1) —C(O)—R—NH—, wherein R typically is —CH(R′)—, wherein R′ is an aminoacid side chain, typically H or a carbon containing substitutent;

or

wherein p is 1, 2, or 3 representing the azetidinecarboxylic acid,proline, or pipecolic acid residues, respectively.

The term “lower” referred to herein in connection with organic radicalssuch as alkyl groups defines such groups with up to and including about6, preferably up to and including 4 and advantageously one or two carbonatoms. Such groups may be straight chain or branched chain.

“Pharmaceutically acceptable salt” includes salts of the compounds ofthe present invention derived from the combination of such compounds andan organic or inorganic acid. In practice the use of the salt formamounts to use of the base form. The compounds of the present inventionare useful in both free base and salt form, with both forms beingconsidered as being within the scope of the present invention.

In addition, the following abbreviations stand for the following:

“ACN” or “CH₃CN” refers to acetonitrile.

“Boc”, “tBoc” or “Tboc” refers to t-butoxy carbonyl.

“DCC” refers to N,N′-dicyclohexylcarbodiimide.

“Fmoc” refers to fluorenylmethoxycarbonyl.

“HBTU” refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphate.

“HOBt” refers to 1-hydroxybenzotriazole monohydrate.

“homoP” or hPro” refers to homoproline.

“MeAla” or “Nme” refers to N-methylalanine.

“naph” refers to naphthylalanine.

“pG” or pGly” refers to pentylglycine.

“tBuG” refers to tertiary-butylglycine.

“ThioP” or tPro” refers to thioproline.

“3Hyp” refers to 3-hydroxyproline

“4Hyp” refers to 4-hydroxyproline

“NAG” refers to N-alkylglycine

“NAPG” refers to N-alkylpentylglycine

“Norval” refers to norvaline

“Norleu” refers to norleucine

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequence for exendin-3 [SEQ. ID. NO. 1].

FIG. 2 depicts the amino acid sequence for exendin-4 [SEQ. ID. NO. 2].

FIG. 3 depicts the amino acid sequences for certain exendin agonistcompounds useful in the present invention [SEQ. ID. NOS. 9 TO 25].

FIG. 4 depicts the amino acid sequences for certain compounds of thepresent invention, Compounds 1-174 [SEQ. ID. NOS. 26-199].

FIG. 5 is a graph showing the effect of functional nephrectomy onexendin-4 clearance.

FIG. 6 is a graph showing the terminal decay of exendin-4 plasma levelsin nephrectomized and sham subjects.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel modified exendins and exendinagonists having an exendin or exendin agonist linked to one or morepolythylene glycol polymers, and related products and methods that areuseful, for example, in the treatment of diabetes, including Type 1,Type 2, and gestational diabetes, in the treatment of disorders whichwould be benefited by agents which modulate plasma glucose levels orsuppress glucagon secretion, and in the treatment of disorders whichwould be benefited by the administration of agents useful in modulatingthe rate of gastric emptying or food intake, including obesity, eatingdisorders, insulin-resistance syndrome, and trigyceride levels, and totreat subjects suffering from dyslipidemia. The methods are also usefulfor lowering plasma lipid levels, reducing cardiac risk, reducingappetite, and reducing the weight of subjects. Pharmaceuticalcompositions for use in the methods of the invention are also disclosed.

Modified Exendins and Exendin Agonists

The modified exendins and exendin agonists of the present inventioninclude one or more PEG polymers linked to an exendin or exendinagonist, such as a naturally occurring exendin, a synthetic exendin oran exendin agonist.

Exendin-4

Exendin-4 is a naturally occurring peptide isolated from the salivarysecretions of the Gila monster. Animal testing of exendin-4 has shownthat its ability to lower blood glucose persists for several hours.Exendin-4, a 39-amino acid polypeptide, is synthesized using solid phasesynthesis as described herein.

As described herein, the nonclinical pharmacology of exendin-4 has beenstudied. In the brain, exendin-4 binds principally to the area postremaand nucleus tractus solitarius region in the hindbrain and to thesubformical organ in the forebrain. Exendin-4 binding has been observedin the rat and mouse brain and kidney. The structures to which exendin-4binds in the kidney are unknown.

Various experiments have compared the biologic actions of exendin-4 andGLP-1 and demonstrated a more favorable spectrum of properties forexendin-4. A single subcutaneous dose of exendin-4 lowered plasmaglucose in db/db (diabetic) and ob/ob (diabetic obese) mice by up to40%. In Diabetic Fatty Zucker (ZDF) rats, 5 weeks of treatment withexendin-4 lowered HbA_(1c) (a measure of glycosylated hemoglobin used toevaluate plasma glucose levels) by up to 41%. Insulin sensitivity wasalso improved by 76% following 5 weeks of treatment in obese ZDF rats.In glucose intolerant primates, dose-dependent decreases in plasmaglucose were also observed.

An insulinotropic action of exendin-4 has also been observed in rodents,improving insulin response to glucose by over 100% in non-fasted HarlanSprague Dawley (HSD) rats, and by up to −10-fold in non-fasted db/dbmice. Higher pretreatment plasma glucose concentrations were associatedwith greater glucose-lowering effects. Thus the observed glucoselowering effect of exendin-4 appears to be glucose-dependent, andminimal if animals are already euglycemic.

Exendin-4 dose dependently slowed gastric emptying in HSD rats and was−90-fold more potent than GLP-1 for this action. Exendin-4 has also beenshown to reduce food intake in NIH/Sw (Swiss) mice following peripheraladministration, and was at least 1000 times more potent than GLP-1 forthis action. Exendin-4 reduced plasma glucagon concentrations byapproximately 40% in anesthetized ZDF rats during hyperinsulinemic,hyperglycemic clamp conditions, but did not affect plasma glucagonconcentrations during euglycemic conditions in normal rats. Exendin-4has been shown to dose-dependently reduce body weight in obese ZDF rats,while in lean ZDF rats, the observed decrease in body weight appears tobe transient.

Through effects on augmenting and restoring insulin secretion, modifiedexendins or exendin agonists containing exendin-4, for example, will beuseful in people with type 2 diabetes who retain the ability to secreteinsulin. Its effects on food intake, gastric emptying, other mechanismsthat modulate nutrient absorption, and glucagon secretion also supportthe utility of such modified exendins and exendin agonists containingexendin-4, for example, in the treatment of, for example, obesity, type1 diabetes, and people with type 2 diabetes who have reduced insulinsecretion.

The toxicology of exendin-4 has been investigated in single-dose studiesin mice, rats and monkeys, repeated-dose (up to 28 consecutive dailydoses) studies in rats and monkeys and in vitro tests for mutagenicityand chromosomal alterations. To date, no deaths have occurred, and therehave been no observed treatment-related changes in hematology, clinicalchemistry, or gross or microscopic tissue changes. Exendin-4 wasdemonstrated to be non-mutagenic, and did not cause chromosomalaberrations at the concentrations tested (up to 5000 μg/mL).

In support of the investigation of the nonclinical pharmacokinetics andmetabolism of exendin-4, a number of immunoassays have been developed. Aradioimmunoassay with limited sensitivity (˜100 pM) was used in initialpharmacokinetic studies. A two-site IRMA assay for exendin-4 wassubsequently validated with a lower limit of quantitation of 15 pM. Thebioavailability of exendin-4, given subcutaneously, was found to beapproximately 50-80% using the radioimmunoassay. This was similar tothat seen following intraperitoneal administration (48-60%). Peak plasmaconcentrations (C_(max)) occurred between 30 and 43 minutes (T_(max)).Both C_(max) and AUC values were monotonically related to dose. Theapparent terminal half-life for exendin-4 given subcutaneously wasapproximately 90-110 minutes. This was significantly longer than the14-41 minutes seen following intravenous dosing. Similar results wereobtained using the IRMA assay. Degradation studies with exendin-4compared to GLP-1 indicate that exendin-4 is relatively resistant todegradation.

Exendin Agonists

Exendin agonists include exendin peptide analogs in which one or morenaturally occurring amino acids are eliminated or replaced with anotheramino acid(s). Preferred exendin agonists are agonist analogs ofexendin-4. Particularly preferred exendin agonists are described incommonly owned PCT Application Serial No. PCT/US98/16387 filed Aug. 6,1998, entitled “Novel Exendin Agonist Compounds,” which claims thebenefit of U.S. Patent Application Ser. No. 60/055,404, filed Aug. 8,1997; commonly owned PCT Application Serial No. PCT/US98/24210, filedNov. 13, 1998, entitled “Novel Exendin Agonist Compounds,” which claimsthe benefit of U.S. Provisional Application No. 60/065,442 filed Nov.14, 1997; and, commonly owned PCT Application Serial No. PCT/US98/24273,filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds,” whichclaims the benefit of U.S. Provisional Application No. 60/066,029 filedNov. 14, 1997, all of which are incorporated herein by reference intheir entirety, including any drawings.

Activity as exendin agonists can be indicated, for example, by activityin the assays described below. Effects of exendins or exendin agonistson gastric motility and gastric emptying can be identified, evaluated,or screened for, using the methods described herein, or other art-knownor equivalent methods for determining gastric motility. For example, seeU.S. patent application Ser. No. 60/166,899, entitled, “High AffinityExendin Receptor,” filed Nov. 22, 1999. Negative receptor assays orscreens for exendin agonist compounds or candidate exendin agonistcompounds, such as an amylin receptor assay/screen using an amylinreceptor preparation as described in U.S. Pat. No. 5,264,372, issuedNov. 23, 1993, the contents of which are incorporated herein byreference, one or more calcitonin receptor assays/screens using, forexample, T47D and MCF7 breast carcinoma cells, which contain calciumreceptors coupled to the stimulation of adenyl cyclase activity, and/ora CGRP receptor assay/screen using, for example, SK-N-MC cells.

One such method for use in identifying or evaluating the ability of acompound to slow gastric motility, involves: (a) bringing together atest sample and a test system, the test sample containing one or moretest compounds, the test system containing a system for evaluatinggastric motility, the system being characterized in that it exhibits,for example, elevated plasma glucose in response to the introduction tothe system of glucose or a meal; and, (b) determining the presence oramount of a rise in plasma glucose in the system. Positive and/ornegative controls may be used as well.

Also included within the scope of the present invention arepharmaceutically acceptable salts of the modified compounds of formula(I-VIII) and pharmaceutical compositions including said compounds andsalts thereof.

Formula I

Exendin agonist compounds also include those described in U.S.Provisional Application No. 60/065,442, including compounds of theformula (I) [SEQ ID NO. 200]:

Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;wherein

Xaa₁ is His, Arg or Tyr; Xaa₂ is Ser, Gly, Ala or Thr; Xaa₂ is Asp orGlu; Xaa₆ is Ala or Thr;

Xaa₆ is Ala, Phe, Tyr or naphthylalanine;

Xaa₇ is Thr or Ser; Xaa₈ is Ala, Ser or Thr; Xaa₉ is Asp or Glu;

Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ isAla or Val; Xaa₂₀ is Ala or Arg; Xaa₂₁ is Ala or Leu;

Xaa₂₂ is Ala, Phe, Tyr or naphthylalanine;Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

Xaa₂₆ is Ala or Leu; Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁ is—OH,

-   -   —NH₂    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly A_(SD-149564.1)Gly Xaa₃₁ Ser Ser Gly        Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;    -   [SEQ ID NO. 201]    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,    -   homoproline, 3Hyp, 4Hyp, thioproline,    -   N-alkylglycine, N-alkylpentylglycine or    -   N-alkylalanine; and    -   Z₂ is —OH or —NH₂;        provided that no more than three of Xaa₃, Xaa₆, Xaa₆, Xaa₈,        Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉,        Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala.        Preferred N-alkyl groups for N-alkylglycine,        N-alkylpentylglycine and N-alkylalanine include lower alkyl        groups preferably of 1 to about 6 carbon atoms, more preferably        of 1 to 4 carbon atoms.

Preferred exendin agonist compounds include those wherein Xaa₁ is His orTyr. More preferably Xaa₁ is His.

Preferred are those compounds wherein Xaa₂ is Gly.

Preferred are those compounds wherein Xaa₁₄ is Leu, pentylglycine orMet.

Preferred compounds are those wherein Xaa₂₅ is Trp or Phe.

Preferred compounds are those where Xaa₆ is Phe or naphthylalanine;Xaa₂₂ is Phe or naphthylalanine and Xaa₂₃ is Ile or Val.

Preferred are compounds wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ areindependently selected from Pro, homoproline, thioproline andN-alkylalanine.

Preferably Z₁ is —NH₂.

Preferably Z₂ is —NH₂.

According to one aspect, preferred are compounds of formula (I) whereinXaa₁ is His or Tyr, more preferably His; Xaa₂ is Gly; Xaa₆ is Phe ornaphthylalanine; Xaa₁₄ is Leu, pentylglycine or Met; Xaa₂₂ is Phe ornaphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ areindependently selected from Pro, homoproline, thioproline orN-alkylalanine. More preferably Z₁ is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (I) wherein: Xaa₁ is His or Arg; Xaa₂is Gly or Ala; Xaa₃ is Asp or Glu; Xaa₆ is Ala or Thr; Xaa₆ is Ala, Pheor nephthylalaine; Xaa₇ is Thr or Ser; Xaa₈ is Ala, Ser or Thr; Xaa₉ isAsp or Glu; Xaa₁₀ is Ala, Leu or pentylglycine; Xaa₁₁ is Ala or Ser;Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln; Xaa₁₄ is Ala, Leu orpentylglycine; Xaa₁₆ is Ala or Glu; Xaa₁₆ is Ala or Glu; Xaa₁₇ is Ala orGlu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala or Arg; Xaa₂₁ is Ala or Leu;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile, Val or tert-butylglycine;Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp or Phe; Xaa₂₆ is Ala or Leu;Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁ is —OH, —NH₂, Gly-Z₂, GlyGly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂, Gly Gly Xaa₃₁ SerSer-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂, Gly GlyXaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂; Xaa₃₁,Xaa₃₆, Xaa₃₇ and Xaa₃₈ being independently Pro homoproline, thioprolineor N-methylalanine; and Z₂ being —OH or —NH₂; provided that no more thanthree of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄,Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ andXaa₂₈ are Ala. Especially preferred compounds include those set forth inPCT application Serial No. PCT/US98/24210, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds” identified therein as compounds 2-23.

According to an especially preferred aspect, provided are compoundswhere Xaa₁₄ is Leu, Ile, Val or pentylglycine, more preferably Leu orpentylglycine, and Xaa₂₅ is Phe, Tyr or naphthylalanine, more preferablyPhe or naphthylalanine. These compounds will be less susceptive tooxidative degration, both in vitro and in vivo, as well as duringsynthesis of the compound.

Formula II

Exendin agonist compounds also include those described in U.S.Provisional Application No. 60/066,029, including compounds of theformula (II) [SEQ ID NO. 202]:

Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉ Xaa₂₀Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;wherein

Xaa₁ is His, Arg, Tyr, Ala, Norval, Val or Norleu; Xaa₂ is Ser, Gly, Alaor Thr; Xaa₃ is Ala, Asp or Glu; Xaa₄ is Ala, Norval, Val, Norleu orGly; Xaa₅ is Ala or Thr;

Xaa₆ is Phe, Tyr or naphthylalanine;

Xaa₇ is Thr or Ser; Xaa₈ is Ala, Ser or Thr; Xaa₉ is Ala, Norval, Val,Norleu, Asp or Glu;

Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ isAla or Val; Xaa₂₀ is Ala or Arg; Xaa₂₃ is Ala or Leu;

Xaa₂₂ is Phe, Tyr or naphthylalanine;Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

Xaa₂₆ is Ala or Leu; Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn;

Z₁ is —OH,

-   -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂;        -   [SEQ ID NO. 203] wherein    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently    -   Pro, homoproline, 3Hyp, 4Hyp, thioproline,    -   N-alkylglycine, N-alkylpentylglycine or    -   N-alkylalanine; and    -   Z₂ is —OH or —NH₂;        provided that no more than three of Xaa₃, Xaa₄, Xaa₅, Xaa₆,        Xaa₈, Xaa₉, Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆,        Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈        are Ala; and provided also that, if Xaa₁ is His, Arg or Tyr,        then at least one of Xaa₃, Xaa₄ and Xaa₉ is Ala.

Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine andN-alkylalanine include lower alkyl groups preferably of 1 to about 6carbon atoms, more preferably of 1 to 4 carbon atoms. Suitable compoundsof formula (II) include those described in application Serial No.PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds”, identified therein in Examples 1-89 (“Compounds 1-89,”respectively), as well as those corresponding compounds identifiedtherein in Examples 104 and 105.

Preferred such exendin agonist compounds include those wherein Xaa₁ isHis, Ala or Norval. More preferably Xaa₁ is His or Ala. Most preferablyXaa₁ is His.

Preferred are those compounds of formula (II) wherein Xaa₂ is Gly.

Preferred are those compounds of formula (II) wherein Xaa₃ is Ala.

Preferred are those compounds of formula (II) wherein Xaa₄ is Ala.

Preferred are those compounds of formula (II) wherein Xaa₉ is Ala.

Preferred are those compounds of formula (II) wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (II) are those wherein Xaa₂₅ is Trp orPhe.

Preferred compounds of formula (II) are those where Xaa₆ is Ala, Phe ornaphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ is Ile orVal.

Preferred are compounds of formula (II) wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ andXaa₃₈ are independently selected from Pro, homoproline, thioproline andN-alkylalanine.

Preferably Z₁ is —NH₂.

Preferably Z₂ is —NH₂.

According to one aspect, preferred are compounds of formula (II) whereinXaa₁ is Ala, His or Tyr, more preferably Ala or His; Xaa₂ is Ala or Gly;Xaa₆ is Phe or naphthylalanine; Xaa₁₄ is Ala, Leu, pentylglycine or Met;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from Pro, homoproline,thioproline or N-alkylalanine; and Xaa₃₉ is Ser or Tyr, more preferablySer. More preferably Z₁ is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (II) wherein: Xaa₁ is His or Ala;Xaa₂ is Gly or Ala; Xaa₃ is Ala, Asp or Glu; Xaa₄ is Ala or Gly; Xaa₅ isAla or Thr; Xaa₆ is Phe or naphthylalanine; Xaa₇ is Thr or Ser; Xaa₈ isAla, Ser or Thr; Xaa₉ is Ala, Asp or Glu; Xaa₁₀ is Ala, Leu orpentylglycine; Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala orGln; Xaa₁₄ is Ala, Leu, Met or pentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala orArg; Xaa₂₁ is Ala or Leu; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile,Val or tert-butylglycine; Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp orPhe; Xaa₂₆ is Ala or Leu; Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁is —OH, —NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, Gly GlyXaa₃₁ Ser Ser Gly Ala-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, GlyGly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈Xaa₃₉-Z₂; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ being independently Prohomoproline, thioproline or N-methylalanine; and Z₂ being —OH or —NH₂;provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and provided also that, ifXaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄ and Xaa₉ isAla. Especially preferred compounds of formula (II) include thosedescribed in application Serial No. PCT/US98/24273, filed Nov. 13, 1998,entitled “Novel Exendin Agonist Compounds” as having the amino acidsequence of SEQ. ID. NOS. 5-93 therein.

According to an especially preferred aspect, provided are compounds offormula (II) where Xaa₁₄ is Ala, Leu, Ile, Val or pentylglycine, morepreferably Leu or pentylglycine, and Xaa₂₅ is Ala, Phe, Tyr ornaphthylalanine, more preferably Phe or naphthylalanine. These compoundswill be less susceptible to oxidative degration, both in vitro and invivo, as well as during synthesis of the compound.

Formula III

Also within the scope of the present invention are narrower genera ofcompounds having peptides of various lengths, for example genera ofcompounds which do not include peptides having a length of 28, 29 or 30amino acid residues, respectively. Additionally, the present inventionincludes narrower genera of compounds described in PCT applicationSerial No. PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel ExendinAgonist Compounds” and having particular amino acid sequences, forexample, compounds of the formula (III) [SEQ. ID. NO. 204]:

Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₈ Xaa₁₉Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;wherein

Xaa₁ is His or Arg; Xaa₂ is Gly or Ala; Xaa₃ is Asp or Glu; Xaa₅ is Alaor Thr;

Xaa₆ is Ala, Phe or naphthylalanine;

Xaa₇ is Thr or Ser; Xaa₈ is Ala, Ser or Thr; Xaa₉ is Asp or Glu;

Xaa₁₀ is Ala, Leu or pentylglycine;

Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu or pentylglycine;

Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ isAla or Val; Xaa₂₀ is Ala or Arg; Xaa₂₁ is Ala or Leu;

Xaa₂₂ is Phe or naphthylalanine;Xaa₂₃ is Ile, Val or tert-butylglycine;

Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp, or Phe; Xaa₂₆ is Ala orLeu; Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁ is —OH,

-   -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or Gly Gly Xaa₃₁        Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from        the group consisting of Pro, homoproline, thioproline and        N-methylylalanine; and    -   Z₂ is —OH or —NH₂;        provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈,        Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉,        Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and        pharmaceutically acceptable salts thereof.

Formula IV

Additionally, the present invention includes narrower genera of peptidecompounds described in PCT Application Serial No. PCT/US98/24273, filedNov. 13, 1998, entitled “Novel Exendin Agonist Compounds” as havingparticular amino acid sequences, for example, compounds of the formula[IV] [SEQ. ID. NO. 205]:

Xaa₁ Xaa₂ Xaa₃ Xaa₅ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₈Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z₁;wherein

Xaa₁ is His or Ala; Xaa₂ is Gly or Ala; Xaa₃ is Ala, Asp or Glu; Xaa₄ isAla or Gly; Xaa₅ is Ala or Thr;

Xaa₆ is Phe or naphthylalanine;

Xaa₇ is Thr or Ser; Xaa₈ is Ala, Ser or Thr; Xaa₉ is Ala, Asp or Glu;

Xaa₁₀ is Ala, Leu or pentylglycine;

Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Met or pentylglycine;

Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ isAla or Val; Xaa₂₀ is Ala or Arg; Xaa₂₁ is Ala or Leu;

Xaa₂₂ is Phe or naphthylalanine;Xaa₂₃ is Ile, Val or tert-butylglycine;

Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp or Phe; Xaa₂₆ is Ala or Leu;Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁ is —OH,

-   -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈    -   Ser-Z₂; [SEQ ID NO. 206]:    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,        homoproline, thioproline, or    -   N-methylylalanine; and    -   Z₂ is —OH or —NH₂;        provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈,        Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₇,        Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇, and Xaa₂₈ are Ala; and        provided that, if Xaa₁ is His, Arg or Tyr, then at least one of        Xaa₃, Xaa₄ and Xaa₉ is Ala; and pharmaceutically acceptable        salts thereof.

Preferred compounds of formula (IV) include those wherein Xaa₁ is His,Ala, Norval or 4-imidazopropionyl. Preferably, Xaa₁ is His, or4-imidazopropionyl or Ala, more preferably His or 4-imidazopropionyl.

Preferred compounds of formula (IV) include those wherein Xaa₂ is Gly.

Preferred compounds of formula (IV) include those wherein Xaa₄ is Ala.

Preferred compounds of formula (IV) include those wherein Xaa₉ is Ala.

Preferred compounds of formula (IV) include those wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (IV) include those wherein Xaa₂₅ is Trpor Phe.

Preferred compounds of formula (IV) include those wherein Xaa₆ is Ala,Phe or naphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ isIle or Val.

Preferred compounds of formula (IV) include those wherein Z₁ is —NH₂.

Preferred compounds of formula (IV) include those wherein Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from the group consisting ofPro, homoproline, thioproline and N-alkylalanine.

Preferred compounds of formula (IV) include those wherein Xaa₃₉ is Seror Tyr, preferably Ser.

Preferred compounds of formula (IV) include those wherein Z₂ is —NH₂.

Preferred compounds of formula (IV) include those wherein Z₁ is —NH₂.

Preferred compounds of formula (IV) include those wherein Xaa₂₁ isLys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straight chain or branchedalkanoyl.

Preferred compounds of formula (IV) include those wherein X₁ is Lys Asn,Lys-NH^(ε)—R Asn, or Lys-NH^(ε)—R Ala where R is Lys, Arg, C₁-C₁₀straight chain or branched alkanoyl. Preferred compounds of formula (IV)include those having an amino acid sequence described in PCT applicationSerial No. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel ExendinAgonist Compounds” as being selected from SEQ. ID. NOS. 95-110 therein.

Formula V

Also provided are compounds described in PCT application PCT/US98/24210,filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds”,including compounds of the formula (V) [SEQ. ID. NO. 207]:

                    5                   10Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ X₁-Z₁;whereinXaa₁ is His, Arg or Tyr or 4-imidazopropionyl;

Xaa₂ is Ser, Gly, Ala or Thr; Xaa₃ is Asp or Glu; Xaa₅ is Ala or Thr;

Xaa₆ is Ala, Phe, Tyr or naphthylalanine;

Xaa₇ is Thr or Ser; Xaa₈ is Ala, Ser or Thr; Xaa₉ is Asp or Glu;

Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ isAla or Val; Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala, Leu or Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straightchain or branched alkanoyl or cycloalkylalkanoyl;Xaa₂₂ is Phe, Tyr or naphthylalanine;Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

Xaa₂₆ is Ala or Leu;

X₁ is Lys Asn, Asn Lys, Lys-NH^(ε)—R Asn, Asn Lys-NH^(ε)—R, Lys-NH^(ε)—RAla, Ala Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straight chain orbranched alkanoyl or cycloalkylalkanoyl

Z₁ is —OH,

-   -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂;        wherein    -   Xaa₃₁, Xaa_(n), Xaa₃₇ and Xaa₃₈ are independently selected from        the group consisting of Pro, homoproline, 3Hyp, 4Hyp,        thioproline, N-alkylglycine, N-alkylpentylglycine and        N-alkylalanine; and    -   Z₂ is —OH or —NH₂;        provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈,        Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉,        Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, and Xaa₂₆ are Ala. Also within the        scope of the present invention are pharmaceutically acceptable        salts of the compound of formula (V) and pharmaceutical        compositions including said compounds and salts thereof.

Preferred exendin agonist compounds of formula (V) include those whereinXaa₁ is His, Tyr or 4-imidazopropionyl. More preferably Xaa₁ is His.

Preferred are those compounds of formula (V) wherein Xaa₁ is4-imidazopropionyl.

Preferred are those compounds of formula (V) wherein Xaa₂ is Gly.

Preferred compounds of formula (V) are those wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (V) are those wherein Xaa₂₅ is Trp orPhe.

According to one aspect, preferred are compounds of formula (V) whereinXaa₆ is Phe or naphthylalanine; and Xaa₂₂ is Phe or naphthylalanine; andXaa₂₃ is Ile or Val. More preferably, Z₁ is —NH₂. According to oneaspect, especially preferred are such compounds of formula (V) whereinXaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from the groupconsisting of Pro, homoproline, thioproline and N-alkylalanine. Morepreferred, Z₂ is —NH₂.

Preferred compounds of formula (V) include those wherein X₁ is Lys Asn,Lys-NH^(ε)—R Asn, or Lys-NH^(ε)—R Ala where R is Lys, Arg, C₁-C₁₀straight chain or branched alkanoyl. Preferred compounds of formula (V)include compounds described in PCT application Serial No.PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds” and identified therein as Compound Nos. 62-69.

Preferred such exendin agonist compounds include those wherein Xaa₁ isHis, Ala or Norval. More preferably Xaa₁ is His or Ala. Most preferablyXaa₁ is His.

Preferred are those compounds of formula (V) wherein Xaa₂ is. Gly.

Preferred are those compounds of formula (V) wherein Xaa₂ is Ala.

Preferred are those compounds of formula (V) wherein Xaa₄ is Ala.

Preferred are those compounds of formula (V) wherein Xaa₉ is Ala.

Preferred are those compounds of formula (V) wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (V) are those wherein Xaa₂₅ is Trp orPhe.

Preferred compounds of formula (V) are those where Xaa₆ is Ala, Phe ornaphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ is Ile orVal.

Preferred are compounds of formula (V) wherein Xaa₃₁, Xaa₃₆, Xaa₃₇ andXaa₃₈ are independently selected from Pro, homoproline, thioproline andN-alkylalanine.

Preferably Z₁ is —NH₂.

Preferably Z₂ is —NH₂.

According to one aspect, preferred are compounds of formula (V) whereinXaa₁ is Ala, His or Tyr, more preferably Ala or His; Xaa₂ is Ala or Gly;Xaa₆ is Phe or naphthylalanine; Xaa₁₄ is Ala, Leu, pentylglycine or Met;Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile or Val; Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from Pro, homoproline,thioproline or N-alkylalanine; and Xaa₃₉ is Ser or Tyr, more preferablySer. More preferably Z₁ is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (V) wherein: Xaa₁ is His or Ala; Xaa₂is Gly or Ala; Xaa₃ is Ala, Asp or Glu; Xaa₄ is Ala or Gly; Xaa_(s) isAla or Thr; Xaa₆ is Phe or naphthylalanine; Xaa₇ is Thr or Ser; Xaa₈ isAla, Ser or Thr; Xaa₉ is Ala, Asp or Glu; Xaa₁₀ is Ala, Leu orpentylglycine; Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala orGln; Xaa₁₄ is Ala, Leu, Met or pentylglycine; Xaa₁₅ is Ala or Glu; Xaa₁₆is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala orArg; Xaa₂₁ is Ala or Leu; Xaa₂₂ is Phe or naphthylalanine; Xaa₂₃ is Ile,Val or tert-butylglycine; Xaa₂₄ is Ala, Glu or Asp; Xaa₂₅ is Ala, Trp orPhe; Xaa₂₆ is Ala or Leu; Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁is —OH, —NH₂, Gly-Z₂, Gly Gly-Z₂, Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly-Z₂, Gly GlyXaa₃₁ Ser Ser Gly Ala-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, GlyGly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly AlaXaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈Xaa₃₉-Z₂; Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ being independently Prohomoproline, thioproline or N-methylalanine; and Z₂ being —OH or —NH₂;provided that no more than three of Xaa₃, Xaa₅, Xaa₆, Xaa₈, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and provided also that, ifXaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄ and Xaa₉ isAla. Especially preferred compounds of formula (V) include thosedescribed in PCT application Serial No. PCT/US98/24210, filed Nov. 13,1998, entitled “Novel Exendin Agonist Compounds” and having the aminoacid sequences identified therein as SEQ. ID. NOS. 5-93.

According to an especially preferred aspect, provided are compounds offormula (V) where Xaa₁₄ is Ala, Leu, Ile, Val or pentylglycine, morepreferably Leu or pentylglycine, and Xaa₂₅ is Ala, Phe, Tyr ornaphthylalanine, more preferably Phe or naphthylalanine. These compoundswill be less susceptible to oxidative degration, both in vitro and invivo, as well as during synthesis of the compound.

Formula VI

Also provided are peptide compounds described in PCT Application SerialNo. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin AgonistCompounds”, including compounds of the formula (VI) [SEQ. ID. NO. 208]:

                    5                   10Xaa₁ Xaa₂ Xaa₃ Gly Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ X₁-Z₁;whereinXaa₁ is His, Arg, Tyr, Ala, Norval, Val, Norleu or 4-imidazopropionyl;

Xaa₂ is Ser, Gly, Ala or Thr; Xaa₃ is Ala, Asp or Glu; Xaa₄ is Ala,Norval, Val, Norleu or Gly; Xaa₅ is Ala or Thr;

Xaa₆ is Phe, Tyr or naphthylalanine;

Xaa₇ is Thr or Ser; Xaa₈ is Ala, Ser or Thr; Xaa₉ is Ala, Norval, Val,Norleu, Asp or Glu;

Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;

Xaa₁₁ is Ala or Ser; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln;

Xaa₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;

Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala or Glu; Xaa₁₇ is Ala or Glu; Xaa₁₉ isAla or Val; Xaa₂₀ is Ala or Arg;

Xaa₂₁ is Ala, Leu or Lys-NH^(ε)—R where R is Lys, Arg, C¹⁻¹⁰ straightchain or branched alkanoyl or cycloalleyl-alkanoyl;Xaa₂₂ is Phe, Tyr or naphthylalanine;Xaa₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;

Xaa₂₄ is Ala, Glu or Asp;

Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;

Xaa₂₆ is Ala or Leu;

X₁ is Lys Asn, Asn Lys, Lys-NH^(ε)—R Asn, Asn Lys-NH^(ε)—R, Lys-NH^(ε)—RAla, Ala Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straight chain orbranched alkanoyl or cycloalkylalkanoyl

Z₁ is —OH,

-   -   —NH₂,    -   Gly-Z₂,    -   Gly Gly-Z₂,    -   Gly Gly Xaa₃₁-Z₂,    -   Gly Gly Xaa₃₁ Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser    -   Gly Gly Xaa₃₁ Ser Ser Gly    -   Gly Gly Xaa_(n) Ser Ser Gly Ala Xaa₃₆-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇-Z₂,    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or    -   Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂;    -   wherein    -   Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently selected from        the group consisting of Pro, homoproline, 3Hyp, 4Hyp,        thioproline, N-alkylglycine, N-alkylpentylglycine and        N-alkylalanine; and    -   Z₂ is —OH or —NH₂;        provided that no more than three of Xaa₃, Xaa₄, Xaa₅, Xaa₆,        Xaa₈, Xaa₉, Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆,        Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁, Xaa₂₄, Xaa₂₅, Xaa₂₆, are Ala; and        provided also that, if Xaa₁ is His, Arg, Tyr, or        4-imidazopropionyl then at least one of Xaa₃, Xaa₄ and Xaa₉ is        Ala.

Preferred compounds of formula (VI) include those wherein Xaa₁ is His,Ala, Norval or 4-imidazopropionyl. Preferably, Xaa₁ is His, or4-imidazopropionyl or Ala, more preferably His or 4-imidazopropionyl.

Preferred compounds of formula (VI) include those wherein Xaa₂ is Gly.

Preferred compounds of formula (VI) include those wherein Xaa₄ is Ala.

Preferred compounds of formula (VI) include those wherein Xaa₉ is Ala.

Preferred compounds of formula (VI) include those wherein Xaa₁₄ is Leu,pentylglycine or Met.

Preferred compounds of formula (VI) include those wherein Xaa₂₅ is Trpor Phe.

Preferred compounds of formula (VI) include those wherein Xaa₆ is Ala,Phe or naphthylalanine; Xaa₂₂ is Phe or naphthylalanine; and Xaa₂₃ isIle or Val.

Preferred compounds of formula (VI) include those wherein Z₁ is —NH₂.

Preferred compounds of formula (VI) include those wherein Xaa₃₁, Xaa₃₆,Xaa₃₇ and Xaa₃₈ are independently selected from the group consisting ofPro, homoproline, thioproline and N-alkylalanine.

Preferred compounds of formula (VI) include those wherein Xaa₃₉ is Seror Tyr, preferably Ser.

Preferred compounds of formula (VI) include those wherein Z₂ is —NH₂.

Preferred compounds of formula (VI) include those 42 wherein Z₁ is —NH₂.

Preferred compounds of formula (VI) include those wherein Xaa₂₁ isLys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straight chain or branchedalkanoyl.

Preferred compounds of formula (VI) include those wherein X₁ is Lys Asn,Lys-NH^(ε)—R Asn, or Lys-NH^(ε)—R Ala where R is Lys, Arg, C₁-C₁₀straight chain or branched alkanoyl.

Preferred compounds of formula (VI) include those described in PCTApplication Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled“Novel Exendin Agonist Compounds” as having an amino acid sequenceselected from those identified therein as SEQ. ID. NOS. 95-110.

Formula VII

Compounds particularly useful according to the present invention areexendin agonist compounds described in U.S. patent application Ser. No.09/003,869, filed Jan. 7, 1998, entitled “Use of Exendins And AgonistsThereof For The Reduction of Food Intake”, including compounds of theformula (VII) [SEQ. ID. NO. 209]:

1                   5                   10Xaa₁ Xaa₂ Xaa₃ Gly Thr Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈                 15                  20Ser Lys Gln Xaa₉ Glu Glu Glu Ala Val Arg Leu                25                   30Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Leu Lys Asn Gly Gly Xaa₁₄             35Ser Ser Gly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈-Zwherein Xaa₁ is His, Arg or Tyr; Xaa₂ is Ser, Gly, Ala or Thr; Xaa₃ isAsp or Glu; Xaa₄ is Phe, Tyr or naphthalanine; Xaa₅ is Thr or Ser; Xaa₆is Ser or Thr; Xaa₇ is Asp or Glu; Xaa₈ is Leu, Ile, Val, pentylglycineor Met; Xaa₉ is Leu, Ile, pentylglycine, Val or Met; Xaa₁₀ is Phe, Tyror naphthalanine; Xaa₁₁ is Ile, Val, Leu, pentylglycine,tert-butylglycine or Met; Xaa₁₂ is Glu or Asp; Xaa₁₃ is Trp, Phe, Tyr,or naphthylalanine; Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently Pro,homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,N-alkylpentylglycine or N-alkylalanine; Xaa₁₈ is Ser, Thr or Tyr; and Zis —OH or —NH₂; with the proviso that the compound does not have theformula of either SEQ. ID. NOS. 1 or 2. Preferred N-alkyl groups forN-alkylglycine, N-alkylpentylglycine and N-alkylalanine include loweralkyl groups preferably of 1 to about 6 carbon atoms, more preferably of1 to 4 carbon atoms. Suitable compounds include those having amino acidsequences of SEQ. ID. NOS. 10 to 40. Also useful in the presentinvention are pharmaceutically acceptable salts of the compounds offormula (VII).

Preferred exendin agonist compounds include those wherein Xaa₁ is His orTyr. More preferably Xaa₁ is His.

Preferred are those compounds wherein Xaa₂ is Gly.

Preferred are those compounds wherein Xaa₉ is Leu, pentylglycine or Met.

Preferred compounds include those wherein Xaa₁₃ is Trp or Phe.

Also preferred are compounds where Xaa₄ is Phe or naphthalanine; Xaa₁₁is Ile or Val and Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independentlyselected from Pro, homoproline, thioproline or N-alkylalanine.Preferably N-alkylalanine has a N-alkyl group of 1 to about 6 carbonatoms.

According to an especially preferred aspect, Xaa₁₅, Xaa₁₆ and Xaa₁₇ arethe same amino acid reside.

Preferred are compounds wherein Xaa₁₈ is Ser or Tyr, more preferablySer.

Preferably Z is —NH₂.

According to one aspect, preferred are compounds of formula (VII)wherein Xaa₁ is His or Tyr, more preferably His; Xaa₂ is Gly; Xaa₄ isPhe or naphthalanine; Xaa₉ is Leu, pentylglycine or Met; Xaa₁₀ is Phe ornaphthalanine; Xaa₁₁ is Ile or Val; Xaa₁₄, Xaa₁₆, Xaa₁₆ and Xaa₁₇ areindependently selected from Pro, homoproline, thioproline orN-alkylalanine; and Xaa₁₆ is Ser or Tyr, more preferably Ser. Morepreferably Z is —NH₂.

According to an especially preferred aspect, especially preferredcompounds include those of formula (VII) wherein: Xaa₁ is His or Arg;Xaa₂ is Gly; Xaa₃ is Asp or Glu; Xaa₄ is Phe or napthylalanine; Xaa₅ isThr or Ser; Xaa₆ is Ser or Thr; Xaa₇ is Asp or Glu; Xaa₆ is Leu orpentylglycine; Xaa₉ is Leu or pentylglycine; Xaa₁₀ is Phe ornaphthylalanine; Xaa₁₁ is Ile, Val or t-butyltylglycine; Xaa₁₂ is Glu orAsp; Xaa₁₃ is Trp or Phe;

Xaa₁₄, Xaa₁₅, Xaa₁₆, and Xaa₁₇ are independently Pro, homoproline,thioproline, or N-methylalanine; Xaa₁₈ is Ser or Tyr: and Z is —OH or—NH₂; with the proviso that the compound does not have the formula ofeither SEQ. ID. NOS. 1 or 2. More preferably Z is —NH₂. Especiallypreferred compounds include those having the amino acid sequence of SEQ.ID. NOS. 10, 11, 22, 23, 24, 27, 29, 36, 37 and 40.

According to an especially preferred aspect, provided are compoundswhere Xaa₉ is Leu, Ile, Val or pentylglycine, more preferably Leu orpentylglycine, and Xaa₁₃ is Phe, Tyr or naphthylalanine, more preferablyPhe or naphthylalanine. These compounds are believed to exhibitadvantageous duration of action and to be less subject to oxidativedegration, both in vitro and in vivo, as well as during synthesis of thecompound.

Formula VIII

Also provided are compounds described in PCT Application Serial No.PCT/US98/16387, filed Aug. 6, 1998, entitled “Novel Exendin AgonistCompounds”, including compounds of the formula (VIII) [SEQ. ID. NO.210]:

1                   5                   10Xaa₁ Xaa₂ Xaa₃ Gly Thr Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈                 15                  20Ser Lys Gln Xaa₉ Glu Glu Glu Ala Val Arg Leu                25                  30Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Leu X₁ Gly Gly Xaa₁₄             35 Ser Ser Gly Ala Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈-Zwherein Xaa₁ is His, Arg, Tyr or 4-imidazopropionyl; Xaa₂ is Ser, Gly,Ala or Thr; Xaa₂ is Asp or Glu; Xaa₄ is Phe, Tyr or naphthylalanine;Xaa₅ is Thr or Ser; Xaa₆ is Ser or Thr; Xaa₇ is Asp or Glu; Xaa₈ is Leu,Ile, Val, pentylglycine or Met; Xaa₉ is Leu, Ile, pentylglycine, Val orMet; Xaa₁₀ is Phe, Tyr or naphthylalanine; Xaa₁₁ is Ile, Val, Leu,pentylglycine, tert-butylglycine or Met; Xaa₁₂ is Glu or Asp; Xaa₁₃ isTrp, Phe, Tyr, or naphthylalanine; X₁ is Lys Asn, Asn Lys, Lys-NH^(ε)—RAsn, Asn Lys-NH^(ε)—R where R is Lys, Arg, C₁-C₁₀ straight chain orbranched alkanoyl or cycloalkylalkanoyl; Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline,N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; Xaa₁₈ is Ser,Thr or Tyr; and Z is —OH or —NH₂; with the proviso that the compounddoes not have the formula of either SEQ. ID. NOS. 1 or 2. Suitablecompounds of formula (VIII) include compounds described in PCTApplication Serial No. PCT/US98/16387, filed Aug. 6, 1998, entitled“Novel Exendin Agonist Compounds” having the amino acid sequences ofSEQ. ID. NOS. 37-40 therein.

Preferred exendin agonist compounds of formula (VIII) include thosewherein Xaa₁ is His, Tyr or 4-imidazopropionyl. More preferably, Xaa₁ isHis or 4-imidazopropionyl.

Preferred are those compounds of formula (VIII) wherein Xaa₂ is Gly.

Preferred are those compounds of formula (VIII) wherein Xaa₉ is Leu,pentylglycine or Met.

Preferred are those compounds of formula (VIII) wherein Xaa₁₃ is Trp orPhe.

Preferred are those compounds of formula (VIII) wherein X₁ is Lys Asn,or Lys-NH^(ε)—R Asn, where R is Lys, Arg, C₁-C₁₀ straight chain orbranched alkanoyl.

Also preferred are compounds of formula (VIII) wherein Xaa₄ is Phe ornaphthylalanine; Xaa₁₀ is Phe or naphthylalanine; Xaa₁₁ is Ile or Valand Xaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently selected from Pro,homoproline, thioproline or N-alkylalanine. According to an especiallypreferred aspect, Xaa₁₈ is Ser or Tyr. Preferred are those suchcompounds wherein Xaa₁₈ is Ser. Preferably, Z is —NH₂.

According to one preferred aspect, preferred are compounds of formula(VIII), wherein Xaa₄ is Phe or naphthylalanine; Xaa₁₀ is Phe ornaphthylalanine; Xaa₁₁ is Ile or Val, X₁ is Lys Asn, or Lys-NH^(ε)—RAsn, where R is Lys, Arg, C₁-C₁₀ straight chain or branched alkanoyl andXaa₁₄, Xaa₁₅, Xaa₁₆ and Xaa₁₇ are independently selected from Pro,homoproline, thioproline or N-alkylalanine.

Preparation of Modified Exendins and Exendin Agonists

The modified exendins and exendin agonists of the present invention maybe made by linking one or more polyethylene glycol polymers or othermolecular weight increasing agents to an exendin or exendin agonist. Thesynthesis of exendins and exendin agonists is thus described first,followed by methodology for linking the polyethylene glycol polymer(s)to the exendin or exendin agonist.

Preparation of Exendins and Exendin Agonists

Exendins and exendin agonist compounds such as exendin analogs andexendin derivatives, described herein may be prepared through peptidepurification as described in, for example, Eng, et al., J. Biol. Chem.265:20259-62, 1990; and Eng, et al., J. Biol. Chem. 267:7402-05, 1992,hereby incorporated by reference herein. Alternatively, exendins andexendin agonist peptides may be prepared by methods known to thoseskilled in the art, for example, as described in Raufman, et al. (J.Biol. Chem. 267:21432-37, 1992), hereby incorporated by referenceherein, using standard solid-phase peptide synthesis techniques andpreferably an automated or semiautomated peptide synthesizer. Thecompounds that constitute active ingredients of the formulations anddosages of the present invention may be prepared using standardsolid-phase peptide synthesis techniques and preferably an automated orsemiautomated peptide synthesizer. Typically, using such techniques, anα-N-carbamoyl protected amino acid and an amino acid attached to thegrowing peptide chain on a resin are coupled at room temperature in aninert solvent such as dimethylformamide, N-methylpyrrolidinone ormethylene chloride in the presence of coupling agents such asdicyclohexylcarbodiimide and 1-hydroxybenzotriazole in the presence of abase such as diisopropylethylamine. The α-N-carbamoyl protecting groupis removed from the resulting peptide-resin using a reagent such astrifluoroacetic acid or piperidine, and the coupling reaction repeatedwith the next desired N-protected amino acid to be added to the peptidechain. Suitable N-protecting groups are well known in the art, witht-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc) beingpreferred herein.

The solvents, amino acid derivatives and 4-methylbenzhydryl-amine resinused in the peptide synthesizer may be purchased from Applied BiosystemsInc. (Foster City, Calif.). The following side chain-protected aminoacids may be purchased from Applied Biosystems, Inc.:BSD-112344.1-Arg(Pmc), Boc-Thr(Bzl), Fmoc-Thr(t-Bu), Boc-Ser(Bzl),Fmoc-Ser(t-Bu), Boc-Tyr(BrZ), Fmoc-Tyr(t-Bu), Boc-Lys(Cl—Z),Fmoc-Lys(Boc), Boc-Glu(Bzl), Fmoc-Glu(t-Bu), Fmoc-His(Trt),Fmoc-Asn(Trt), and Fmoc-Gln(Trt). Boc-His(BOM) may be purchased fromApplied Biosystems, Inc. or Bachem Inc. (Torrance, Calif.). Anisole,dimethylsulfide, phenol, ethanedithiol, and thioanisole may be obtainedfrom Aldrich Chemical Company (Milwaukee, Wis.). Air Products andChemicals (Allentown, Pa.) supplies HF. Ethyl ether, acetic acid andmethanol may be purchased from Fisher Scientific (Pittsburgh, Pa.).

Solid phase peptide synthesis may be carried out with an automaticpeptide synthesizer (Model 430A, Applied Biosystems Inc., Foster City,Calif.) using the NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry(see, Applied Biosystems User's Manual for the ABI 430A PeptideSynthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70, AppliedBiosystems, Inc., Foster City, Calif.) with capping. Boc-peptide-resinsmay be cleaved with HF (−50° C. to 0° C., 1 hour). The peptide may beextracted from the resin with alternating water and acetic acid, and thefiltrates lyophilized. The Fmoc-peptide resins may be cleaved accordingto standard methods (Introduction to Cleavage Techniques, AppliedBiosystems, Inc., 1990, pp. 6-12). Peptides may also be assembled usingan Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).

Peptides may be purified by RP-HPLC (preparative and analytical) using aWaters Delta Prep 3000 system. A C4, C8 or C18 preparative column (10μ,2.2×25 cm; Vydac, Hesperia, Calif.) may be used to isolate peptides, andpurity may be determined using a C4, C8 or C18 analytical column (5μ,0.46×25 cm; Vydac). Solvents (A=0.1% TFA/water and B=0.1% TFA/CH₃CN) maybe delivered to the analytical column at a flowrate of 1.0 ml/min and tothe preparative column at 15 ml/min. Amino acid analyses may beperformed on the Waters Pico Tag system and processed using the Maximaprogram. Peptides may be hydrolyzed by vapor-phase acid hydrolysis (115°C., 20-24 h). Hydrolysates may be derivatized and analyzed by standardmethods (Cohen, et al., The Pico Tag Method: A Manual of AdvancedTechniques for Amino Acid Analysis, pp. 11-52, Millipore Corporation,Milford, Mass. (1989)). Fast atom bombardment analysis may be carriedout by M-Scan, Incorporated (West Chester, Pa.). Mass calibration may beperformed using cesium iodide or cesium iodide/glycerol. Plasmadesorption ionization analysis using time of flight detection may becarried out on an Applied Biosystems Bio-Ion 20 mass spectrometer.Electrospray mass spectroscopy may be carried and on a VG-Trio machine.

Peptide active ingredient compounds useful in the formulations anddosages of the invention may also be prepared using recombinant DNAtechniques, using methods now known in the art. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor(1989). Alternatively, such compounds may be prepared by homogeneousphase peptide synthesis methods. Non-peptide compounds useful in thepresent invention may be prepared by art-known methods. For example,phosphate-containing amino acids and peptides containing such aminoacids, may be prepared using methods known in the art. See, e.g.,Bartlett and Landen, Biorg. Chem. 14:356-377 (1986).

Conjugation of Polyethylene Glycol Polymers (or Other Molecular WeightIncreasing Agents)

There are several strategies for coupling PEG to peptides/proteins. See,Int. J. Hematology 68:1 (1998); Bioconjugate Chem. 6:150 (1995); andCrit. Rev. Therap. Drug Carrier Sys. 9:249 (1992) all of which areincorporated herein by reference in their entirety. Those skilled in theart, therefore, will be able to utilize such well known techniques forlinking one or more polethylene glycol polymers to the exendins andexendin agonists described herein. Suitable polethylene glycol polymerstypically are commercially available or may be made by techniques wellknown to those skilled in the art. The polyethylene glycol polymers orother molecular weight increasing agents preferably have molecularweights between 500 and 20,000 and may be branched or straight chainpolymers.

The attachment of a PEG on an intact peptide or protein can beaccomplished by coupling to amino, carboxyl or thiol groups. Thesegroups will typically be the N and C termini and on the side chains ofsuch naturally occurring amino acids as lysine, aspartic acid, glutamicacid and cysteine. Since exendin-4 and other exendins and exendinagonists can be prepared by solid phase peptide chemistry techniques, avariety of moieties containing diamino and dicarboxylic groups withorthogonal protecting groups can be introduced for conjugation to PEG.

The present invention also provides for conjugation of an exendin orexendin agonist to one or more polymers other than polyethylene glycolwhich can regulate kidney clearance in a manner similar to polyethyleneglycol. Examples of such polymers include albumin and gelatin. See,Gombotz and Pettit, Bioconjugate Chem., 6:332-351, 1995, which isincorporated herein by reference in its entirety.

Utility

The formulations and dosages described herein are useful in view oftheir pharmacological properties. In particular, the compounds of theinvention possess activity as agents to reduce food intake and as agentsto regulate gastric motility and to slow gastric emptying, as evidencedby the ability to inhibit gastric emptying levels in mammals. They canbe used to treat conditions or diseases which can be alleviated byreducing food intake or regulating gastric motility. The formulationsand dosages of the invention are also effective as exendins and exendinagonists, and possess activity as agents to lower blood glucose, and toregulate gastric motility and to slow gastric emptying, as evidenced bythe ability to reduce post-prandial glucose levels in mammals. Thecompounds of the present invention are useful in in vitro and in vivoscientific methods for investigation of exendins and exendin agonistsfor example in methods such as those described herein.

The compounds referenced above may form salts with various inorganic andorganic acids and bases. Such salts include salts prepared with organicand inorganic acids, for example, HCl, HBr, H₂SO₄, H₃PO₄,trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid,toluenesulfonic acid, maleic acid, fumaric acid and camphorsulfonicacid. Salts prepared with bases include ammonium salts, alkali metalsalts, e.g., sodium and potassium salts, and alkali earth salts, e.g.,calcium and magnesium salts. Acetate, hydrochloride, andtrifluoroacetate salts are preferred. The salts may be formed byconventional means, as by reacting the free acid or base forms of theproduct with one or more equivalents of the appropriate base or acid ina solvent or medium in which the salt is insoluble, or in a solvent suchas water which is then removed in vacuo or by freeze-drying or byexchanging the ions of an existing salt for another ion on a suitableion exchange resin.

Formulation and Administration

Modified exendin and exendin agonist formulations and dosages of theinvention are useful in view of their exendin-like effects, and mayconveniently be provided in the form of formulations suitable forparenteral (including intravenous, intramuscular and subcutaneous)administration. Also described herein are formulations and dosagesuseful in alternative delivery routes, including oral, nasal, buccal,sublingual and pulmonary.

The feasibility of alternate routes of delivery for exendin-4 has beenexplored by measuring exendin-4 in the circulation in conjunction withobservation of a biologic response, such as plasma glucose lowering indiabetic animals, after administration. Passage of exendin-4 has beeninvestigated across several surfaces, the respiratory tract (nasal,tracheal and pulmonary routes) and the gut (sublingual, gavage andintraduodenal routes). Biologic effect and appearance of exendin-4 inblood have been observed with each route of administration via therespiratory tract, and with sublingual and gavaged peptide via thegastrointestinal tract. Intra-tracheal administration, nasaladministration, administration via the gut, and sublingualadministration have all been described.

In some cases, it will be convenient to provide a modified exendin orexendin agonist and another anti-gastric-emptying agent, such asglucagon, an amylin, or an amylin agonist, in a single composition orsolution for administration together. In other cases, it may be moreadvantageous to administer another anti-emptying agent separately fromthe modified exendin or exendin agonist. In yet other cases, it may bebeneficial to provide a modified exendin or exendin agonist eitherco-formulated or separately with other glucose lowering agents such asinsulin. A suitable administration format may best be determined by amedical practitioner for each patient individually. Suitablepharmaceutically acceptable carriers and their formulation are describedin standard formulation treatises, e.g., Remington's PharmaceuticalSciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A.“Parenteral Formulations of Proteins and Peptides: Stability andStabilizers,” Journal of Parenteral Science and Technology, TechnicalReport No. 10, Supp. 42:2 S (1988).

Compounds useful in the invention can be provided as parenteralcompositions for injection or infusion. They can, for example, besuspended in an inert oil, suitably a vegetable oil such as sesame,peanut, olive oil, or other acceptable carrier. Preferably, they aresuspended in an aqueous carrier, for example, in an isotonic buffersolution at a pH of about 4.0 to about 7.4. These compositions may besterilized by conventional sterilization techniques, or may be sterilefiltered. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions; such as pH buffering agents. Useful buffers include forexample, sodium acetate/acetic acid buffers. A form of repository or“depot” slow release preparation may be used so that therapeuticallyeffective amounts of the preparation are delivered into the bloodstreamover many hours or days following transdermal injection or delivery.

The desired isotonicity may be accomplished using sodium chloride orother pharmaceutically acceptable agents such as dextrose, boric acid,sodium tartrate, propylene glycol, polyols (such as mannitol andsorbitol), or other inorganic or organic solutes. Sodium chloride ispreferred particularly for buffers containing sodium ions.

The claimed compounds can also be formulated as pharmaceuticallyacceptable salts (e.g., acid addition salts) and/or complexes thereof.Pharmaceutically acceptable salts are non-toxic salts at theconcentration at which they are administered. The preparation of suchsalts can facilitate the pharmacological use by altering thephysical-chemical characteristics of the composition without preventingthe composition from exerting its physiological effect. Examples ofuseful alterations in physical properties include lowering the meltingpoint to facilitate transmucosal administration and increasing thesolubility to facilitate the administration of higher concentrations ofthe drug.

Pharmaceutically acceptable salts include acid addition salts such asthose containing sulfate, hydrochloride, phosphate, sulfamate, acetate,citrate, lactate, tartrate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate.Pharmaceutically acceptable salts can be obtained from acids such ashydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, aceticacid, citric acid, lactic acid, tartaric acid, malonic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid. Suchsalts may be prepared by, for example, reacting the free acid or baseforms of the product with one or more equivalents of the appropriatebase or acid in a solvent or medium in which the salt is insoluble, orin a solvent such as water which is then removed in vacuo or byfreeze-drying or by exchanging the ions of an existing salt for anotherion on a suitable ion exchange resin.

Carriers or excipients can also be used to facilitate administration ofthe compound. Examples of carriers and excipients include calciumcarbonate, calcium phosphate, various sugars such as lactose, glucose,or sucrose, or types of starch, cellulose derivatives, gelatin,vegetable oils, polyethylene glycols and physiologically compatiblesolvents. The compositions or pharmaceutical composition can beadministered by different routes including intravenously,intraperitoneal, subcutaneous, and intramuscular, orally, topically, ortransmucosally.

If desired, solutions of the above compositions may be thickened with athickening agent such as methyl cellulose. They may be prepared inemulsified form, either water in oil or oil in water. Any of a widevariety of pharmaceutically acceptable emulsifying agents may beemployed including, for example, acacia powder, a non-ionic surfactant(such as a Tween), or an ionic surfactant (such as alkali polyetheralcohol sulfates or sulfonates, e.g., a Triton).

Compositions useful in the invention are prepared by mixing theingredients following generally accepted procedures. For example, theselected components may be simply mixed in a blender or other standarddevice to produce a concentrated mixture which may then be adjusted tothe final concentration and viscosity by the addition of water orthickening agent and possibly a buffer to control pH or an additionalsolute to control tonicity.

For use by the physician, the compounds will be provided in dosage unitform containing an amount of an exendin agonist, with or without anotheranti-emptying agent. Therapeutically effective amounts of an exendinagonist for use in the control of gastric, emptying and in conditions inwhich gastric emptying is beneficially slowed or regulated are thosethat decrease post-prandial blood glucose levels, preferably to no morethan about 8 or 9 mM or such that blood glucose levels are reduced asdesired. In diabetic or glucose intolerant individuals, plasma glucoselevels are higher than in normal individuals. In such individuals,beneficial reduction or “smoothing” of post-prandial blood glucoselevels, may be obtained. As will be recognized by those in the field, aneffective amount of therapeutic agent will vary with many factorsincluding the age and weight of the patient, the patient's physicalcondition, the blood sugar level or level of inhibition of gastricemptying to be obtained, and other factors.

Such pharmaceutical compositions are useful in causing gastrichypomotility in a subject and may be used as well in other disorderswhere gastric motility is beneficially reduced.

The effective daily anti-emptying dose of the compounds will typicallybe in the range of 0.01 or 0.03 to about 5 mg/day, preferably about 0.01or 0.5 to 2 mg/day and more preferably about 0.01 or 0.1 to 1 mg/day,for a 70 kg patient, administered in a single or divided doses. Theexact dose to be administered is determined by the attending clinicianand is dependent upon where the particular compound lies within theabove quoted range, as well as upon the age, weight and condition of theindividual. Administration should begin at the first sign of symptoms orshortly after diagnosis of diabetes mellitus. Administration may be byinjection, preferably subcutaneous or intramuscular. Orally activecompounds may be taken orally, however dosages should be increased 5-10fold.

Generally, in treating or preventing elevated, inappropriate, orundesired post-prandial blood glucose levels, the compounds of thisinvention may be administered to patients in need of such treatment in adosage ranges similar to those given above, however, the compounds areadministered more frequently, for example, one, two, or three times aday.

The optimal formulation and mode of administration of compounds of thepresent application to a patient depend on factors known in the art suchas the particular disease or disorder, the desired effect, and the typeof patient. While the compounds will typically be used to treat humanpatients, they may also be used to treat similar or identical diseasesin other vertebrates such as other primates, farm animals such as swine,cattle and poultry, and sports animals and pets such as horses, dogs andcats.

To assist in understanding the present invention the following Examplesare included which describe the results of a series of experiments. Theexperiments relating to this invention should not, of course, beconstrued as specifically limiting the invention and such variations ofthe invention, now known or later developed, which would be within thepurview of one skilled in the art are considered to fall within thescope of the invention as described herein and hereinafter claimed.

Example 1 Preparation of Exendin-3

His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala ProPro Pro Ser-NH₂ [SEQ. ID. NO. 1]

The above amidated peptide was assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.). In general, single-coupling cycles were usedthroughout the synthesis and Fast Moc (HBTU activation) chemistry wasemployed. Deprotection (Fmoc group removal) of the growing peptide chainwas achieved using piperidine. Final deprotection of the completedpeptide resin was achieved using a mixture of triethylsilane (0.2 mL),ethanedithiol (0.2 mL), anisole (0.2 mL), water (0.2 mL) andtrifluoroacetic acid (15 mL) according to standard methods (Introductionto Cleavage Techniques, Applied Biosystems, Inc.) The peptide wasprecipitated in ether/water (50 mL) and centrifuged. The precipitate wasreconstituted in glacial acetic acid and lyophilized. The lyophilizedpeptide was dissolved in water). Crude purity was about 75%.

Used in purification steps and analysis were Solvent A (0.1% TFA inwater) and Solvent B (0.1% TFA in ACN).

The solution containing peptide was applied to a preparative C-18 columnand purified (10% to 40% Solvent B in Solvent A over 40 minutes). Purityof fractions was determined isocratically using a C-18 analyticalcolumn. Pure fractions were pooled furnishing the above-identifiedpeptide. Analytical RP-HPLC (gradient 30% to 60% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide gave product peptide havingan observed retention time of 19.2 minutes.

Example 2 Preparation of Exendin-4

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala ProPro Pro Ser-NH₂ [SEQ. ID. NO. 2]

The above amidated peptide was assembled on4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucineMBHA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids(Applied Biosystems, Inc.), cleaved from the resin, deprotected andpurified in a similar way to Exendin-3 as describe in Example 1. Used inanalysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA inACN). Analytical RP-HPLC (gradient 36% to 46% Solvent B in Solvent Aover 30 minutes) of the lyophilized peptide gave product peptide havingan observed retention time of 14.9 minutes. Electrospray MassSpectrometry (M): calculated 4186.6; found 4186.0 to 4186.8 (four lots).

Example 3 Clearance by the Kidney

The kidney can play a major role in the elimination of some molecules(drugs, peptides, proteins). For some molecules, this process beginswhen the kidney filters the blood at the glomerulus to produce theultrafiltrate described below. The glomerular filter discriminates notonly on the basis of molecular weight but also by acting as a negativelycharged selective barrier, promoting retention of anionic compounds. Thefree fraction of molecules in the plasma (not protein bound) with amolecular weight less than 5 kD and an effective radii less than 15 Åare easily filtered. For larger molecular weight molecules they arefiltered on a more restrictive and limited basis, principally bymolecular size, structure and net charge. The cutoff point forglomerular filtration lies between albumin (67 kD) which is retained andhemoglobin (68 kD) which is filtered. Albumin, with an effective radiusof about 36 Å is filtered less than 1% at the glomerulus.

Once in the glomerulus a molecule travels to the proximal tubule whereit is either reabsorbed or it passes on through the loop of Henle to thedistal tubule where collecting ducts drain the filtrate into thebladder. Filtered proteins and peptides are typically cleaved by brushborder enzymes in the proximal tubule, from where they are efficientlyretrieved by sodium/amino cotransporters (scavenging pumps). Otherwise,molecules which are polar, ionized and of large molecular weight willnot be reabsorbed. Throughout this process metabolizing enzymes in therenal cortex (proximal tubules) may also degrade the molecule into morepolar molecules, thereby increasing the probability for excretion intothe urine. Many peptide hormones (for example, amylin, calcitonins, andGLP-1) are degraded by passage through the renal circulation, presumablyby vascular ectoenzymes accessible to the plasma, independently of theprocess of glomerular filtration. In those examples, rates of peptideclearance from the plasma are similar to the rate of renal plasma flow,which is −3-fold greater than the rate of glomerular filtration.

To test whether renal filtration could be the principal mode of exendinelimination, studies were performed in overnight fasted nephrectomizedmale rats infused with exendin-4 at a constant rate. Steady-state plasmalevels of exendin-4 were greatly increased in nephrectomized ratscompared to rats with their kidneys intact. This data indicated that thekidney was responsible for at least 80% of the clearance of exendin-4(see FIGS. 5 and 6). Exendin-4 clearance rates in intact rats weresimilar to glomerular filtration rates expected in those rats (4.2mL/min). Taken together these results indicate that very littlemetabolism seems to occur systemically and that most of the clearance ofexendin-4 is through the kidney via filtration (but not by renalintravascular proteolysis). The low amounts of immunoreactivefull-length exendin-4 in the urine are consistent with it being cleavedby brush border enzymes in the proximal tubule after filtration. Theseresults are also consistent with the fact that studies performed toidentify plasma circulating metabolites of exendin-4 yielded very littleevidence of proteolytic degradation; following large intravenous dosesin animals, HPLC analysis of plasma showed only the presence of intactexendin, and negligible appearance of “daughter” peaks indicative of thebuildup of degradation products. This is in contrast to other peptidesstudied (for example amylin and GLP-1), where the disappearance of the“parent” HPLC peak was associated with the appearance of “daughter” HPLCpeaks, subsequently identified as subpeptide degradants.

Example 4 PEG Modified Exendin-4

Different spectra of biological activities of exendin-4 may be selectedby putting a PEG group at appropriate positions. Loss or alteration ofbioactivity has been reported for PEGylated proteins which may be due tothe presence of the PEG chains themselves, the particular site occupiedby the PEG chain, or the coupling conditions having an adverse effect onthe protein.

Primary considerations for PEG modification in terms of filtration atthe kidney of exendin and exendin agonists are size and charge.Unmodified, exendin-4 has a molecular weight of approximately 4.2 kD andis anionic in nature with an overall net charge of approximately −2 atphysiological pH. One to ten, preferably one, two or three PEGconstituents may be covalently linked to exendin-4 or an analog ofexendin-4, for example, with one PEG constituent being preferred. Thesize of each independent PEG constituent can vary from a molecularweight of 500 to 20,000, preferably between 5,000 and 12,000.

Many of the methods for covalent attachment of PEG involve theepsilon-amino group on lysine. Exendin-4 has two lysines that could bemodified by attachment of PEG (see compounds 201 and 202, below). Inaddition, the epsilon-amino groups at these positions may be masked,thereby increasing the anionic nature of the peptide.

(201) [SEQ ID NO. 211]: HGEGTFTSDLSK(PEG)QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂(202) [SEQ ID NO. 212]: HGEGTFTSDLSKQMEEEAVRLFIEWLK(PEG)NGGPSSGAPPPS-NH₂

Other positions that may be modified by substitution of a Lys-PEG orequivalent, for example, are:

(203) [SEQ ID NO. 213]: HK(PEG)EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂(204) [SEQ ID NO. 214]: HGEGK(PEG)FTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂(205) [SEQ ID NO. 215]: HGEGTFTK(PEG)DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂(206) [SEQ ID NO. 216]: HGEGTFTSDK(PEG)SKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂(207) [SEQ ID NO. 217]: HGEGTFTSDLK(PEG)KQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂(208) [SEQ ID NO. 218]: HGEGTFTSDLSKK(PEG)MEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂(209) [SEQ ID NO. 219]:*HGEGTFTSDLSKQMEK(PEG)EAVRLFIEWLKNGGPSSGAPPPS-NH₂(210) [SEQ ID NO. 220]:*HGEGTFTSDLSKQMEEK(PEG)AVRLFIEWLKNGGPSSGAPPPS-NH₂ (211) [SEQ ID NO. 221]:HGEGTFTSDLSKQMEEEAK(PEG)RLFIEWLKNGGPSSGAPPPS-NH₂ (212) [SEQ ID NO. 222]:HGEGTFTSDLSKQMEEEAVRK(PEG)FIEWLKNGGPSSGAPPPS-NH₂(213) [SEQ ID NO. 223]:*HGEGTFTSDLSKQMEEEAVRLFIK(PEG)WLKNGGPSSGAPPPS-NH₂ (214) [SEQ ID NO. 224]:HGEGTFTSDLSKQMEEEAVRLFIEK(PEG)LKNGGPSSGAPPPS-NH₂ (215) [SEQ ID NO. 225]:HGEGTFTSDLSKQMEEEAVRLFIEWLKK(PEG)GGPSSGAPPPS-NH₂

The three molecules marked with an asterisk above contain a PEGylatedLys residue substituted for a glutamic acid at the specified location.Those in the art will appreciate that non-K(PEG) substituted moleculesat these positions can instead be modified by conjugation of a PEGmoiety to the glutamic side chain carboxyl group, which modification isreferred to herein as E(PEG).

Other analogs in which Lys-PEG can be substituted include:

(216) [SEQ ID NO. 226]: HGEGTFTSDLSKQMEEEAVRLFIEWLKNK(PEG)GPSSGAPPPS-NH₂(217) [SEQ ID NO. 227]: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGK(PEG)PSSGAPPPS-NH₂

Various molecules, including K(PEG) modified and arginine substitutedexendins, used in Examples 5-10 are shown in Table I, below.

TABLE I exendin-4 [SEQ ID NO. 2] HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (218) [SEQ ID NO. 228] (CH3)-COHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂ (219) [SEQ ID NO. 229](CH3)-CH2HGEGTFTSDLSKQMEEEAVRLFIEWLK NGGPSSGAPPS-NH₂ (220)[SEQ ID NO. 230] HGEGTFTSDRQMEEEAVRLFIEWLK(PEG)NGGPSS GAPPPS-NH₂ (221)[SEQ ID NO. 231] HGEGTFTSDLSK(PEG)QMEEEAVRLFIEWLRNGGP SSGAPPPS-NH₂ (222)[SEQ ID NO. 232] HGEGTFTSDLSRQMEEEAVRLFIEWLRNGGPSSGAP PPS-NH₂ (223)[SEQ ID NO. 233] HGEGTFTSDLSRQMEEEAVRLFIEWLRNGGPSSGAP PPK(PEG)-NH₂ (224)[SEQ ID NO. 234] HGEGTFTSDLSRQMEEEAVRLFIEWLRNGK(PEG)P SSGAPPPS-NH₂ (225)[SEQ ID NO. 235] HGEGTFTSDLSRQMEEEAVRLFIEWLK(PEG)NGGP SSGAPPPS-NH₂ (226)[SEQ ID NO. 236] HGEGTFTSDLSK(PEG)QMEEEAVRLFIEWLRNGGP SSGAPPPS-NH₂ (227)[SEQ ID NO. 237] (PEG)COHGEGTFTSDLSRQMEEEAVRLFIEWLRNG GPSSGAPPPS-NH₂(228) [SEQ ID NO. 238] (PEG)CH2HGEGTFTSDLSRQMEEEAVRLFIEWLRNGGPSSGAPPPS-NH₂ (229) [SEQ ID NO. 239]HGEGTFTSDLSRQMEEAVRLFIEWLRNGGPSSGAPP PK(PEG)-NH₂ (230) [SEQ ID NO. 240]HGEGTFTSDLSRQMEEEAVRLFIEWLRNGK(PEG)P SSGAPPPS-NH₂

The various PEG modified exendins used in Examples 5-10, below, areprovided in Table I, with the corresponding results being provided inTable II (see the end of Example 9). GLP-1 [7-36]NH₂ (GLP-1) waspurchased from Bachem (Torrance, Calif.). All other peptides wereprepared using synthesis methods such as those described herein. Allchemicals were of the highest commercial grade. The cAMP SPA immunoassaywas purchased from Amersham. The radioligands were purchased from NewEngland Nuclear (Boston, Mass.). RINm5f cells (American Type TissueCollection, Rockville, Md.) were grown in DME/F12 medium containing 10%fetal bovine serum and 2 mM L-glutamine. Cells were grown at 37° C. and5% CO₂/95% humidified air and medium was replaced every 2 to 3 days.Cells were grown to confluence then harvested and homogenized using on aPolytron homogenizer. Cell homogenates were stored frozen at −70° C.until used.

Example 5 GLP-1 Receptor Binding Studies

Receptor binding can be assessed by measuring displacement of [¹²⁵I]GLP-1 or [¹²⁵I] exendin (9-39) from RINm5f membranes. Assay buffercontained 5 μg/ml bestatin, 1 μg/ml phosphoramidon, 1 mg/ml bovine serumalbumin (fraction V), 1 mg/ml bacitracin, and 1 mM MgCl₂ in 20 mM HEPES,pH 7.4. To measure binding, 30 μg membrane protein (Bradford proteinassay) is resuspended in 200 μl assay buffer and incubated with 60 pM[¹²⁵I] GLP-1 or [¹²⁵I] exendin(9-39) and unlabeled peptides for 120minutes at 23° C. in 96 well plates (Nagle Nunc, Rochester, N.Y.).Incubations are terminated by rapid filtration with cold phosphatebuffered saline, pH 7.4, through polyethyleneimine-treated GF/B glassfiber filters (Wallac Inc., Gaithersburg, Md.) using a Tomtec Mach IIplate harvester (Wallac Inc., Gaithersburg, Md.). Filters are dried,combined with scintillant, and radioactivity determined in a Betaplateliquid scintillant counter (Wallac Inc.).

Peptide samples are run in the assay as duplicate points at 6 dilutionsover a concentration range of 10⁻⁶M to 10⁻¹²M to generate responsecurves. The biological activity of a sample can be expressed as an IC₅₀value, calculated from the raw data using an iterative curve-fittingprogram using a 4-parameter logistic equation (Prizm, GraphPADSoftware).

Example 6 Cyclase Activation Study

Assay buffer contained 10 μM GTP, 0.75 mM ΔTP, 2.5 mM MgCl₂, 0.5 mMphosphocreatine, 12.5 U/ml creatine kinase, 0.4 mg/ml aprotinin, 1 μMIBMX in 50 mM HEPES, pH 7.4. Membranes and peptides was combined in 100ml of assay buffer in 96 well filter-bottom plates (Millipore Corp.,Bedford, Mass.). After 20 minutes incubation at 37° C., the assay wasterminated by transfer of supernatant by filtration into a fresh 96 wellplate using a Millipore vacuum manifold. Supernatant cAMP contents werequantitated by SPA immunoassay. Peptide samples were run in the assay astriplicate points at 7 dilutions over a concentration range of 10⁻⁶M to10⁻¹²M to generate response curves. The biological activity of aparticular sample was expressed as an EC₅₀ value calculated as describedabove.

Example 7 Determination of Blood Glucose Levels in db/db Mice

C57BLKS/J-m-db mice at least 3 months of age are utilized for the study.The mice can be obtained from The Jackson Laboratory and allowed toacclimate for at least one week before use. Mice can be housed in groupsof ten at 22° C.±1° C. with a 12:12 light:dark cycle, with lights on at6 a.m. All animals can be deprived of food for 2 hours before takingbaseline blood samples. Approximately 70 μl of blood is drawn from eachmouse via eye puncture, after a light anesthesia with metophane. Aftercollecting baseline blood samples, to measure plasma glucoseconcentrations, all animals receive subcutaneous injections of eithervehicle (10.9% NaCl), exendin-4 or test compound (1 μg) in vehicle.Blood samples were drawn again, using the same procedure, after exactlyone hour from the injections, and plasma glucose concentrations weremeasured. For each animal, the % change in plasma value, from baselinevalue, was calculated.

Example 8 Dose Response Determination of Blood Glucose Levels in db/dbMice

C57BLKS/J-m-db/db mice, at least 3 months of age, were utilized. Themice were obtained from The Jackson Laboratory and allowed to acclimatefor at least one week before use. Mice were housed in groups of ten at22° C.±1° C. with a 12:12 light:dark cycle, with lights on at 6 a.m. Allanimals were deprived of food for 2 hours before taking baseline bloodsamples. Approximately 70 μl of blood was drawn from each mouse via eyepuncture, after a light anesthesia with metophane. After collectingbaseline blood samples, to measure plasma glucose concentrations, allanimals receive subcutaneous injections of either vehicle, exendin-4 ortest compound. Blood samples were drawn again, using the same procedure,after exactly one hour from the injections, and plasma glucoseconcentrations were measured. For each animal, the % change in plasmavalue, from baseline value, was calculated and a dose dependentrelationship was evaluated using Graphpad Prizm™ software.

Example 9 Gastric Emptying

A gastric emptying study may also be carried out to examine the effectsof exendin-4 and/or an exendin agonist compound on gastric emptying inrats. Such experiments typically follow a modification of the method ofScarpignato, et al., Arch. Int. Pharmacodyn. Ther. 246:286-94, 1980.Male Harlan Sprague Dawley (HSD) rats are used. All animals are housedat 22.7±0.8° C. in a 12:12 hour light:dark cycle (experiments beingperformed during the light cycle) and were fed and watered ad libitum(Diet LM-485, Teklad, Madison, Wis.). The determination of gastricemptying by the method described below can be performed after a fast of−20 hours to ensure that the stomach contained no chyme that wouldinterfere with spectrophotometric absorbance measurements.

Conscious rats receive by gavage 1.5 ml of an acaloric gel containing1.5% methyl cellulose (M-0262, Sigma Chemical Co, St Louis, Mo.) and0.05% phenol red indicator. Twenty minutes after gavage, rats areanesthetized using 5% halothane, the stomach is exposed and clamped atthe pyloric and lower esophageal sphincters using artery forceps,removed and opened into an alkaline solution made up to a fixed volume.Stomach content is derived from the intensity of the phenol red in thealkaline solution, measured by absorbance at a wavelength of 560 nm. Inseparate experiments on several other rats, the stomach and smallintestine can be both excised and opened into an alkaline solution. Thequantity of phenol red that could be recovered from the uppergastrointestinal tract within 20 minutes of gavage can then bedetermined. Dye which appears to bind irrecoverably to the gut luminalsurface accounts for the balance. To account for a maximal dye recoveryof less than 100%, the percentage of stomach contents remaining after 20min. are expressed as a fraction of the gastric contents recovered fromcontrol rats sacrificed immediately after gavage in the same experiment.Percent gastric contents remaining=(absorbance at 20 min)/(absorbance at0 mm)×100.

Example 10 Test Compound Injections Reduced Food Intake in Normal Mice

All mice (NIH:Swiss mice) were housed in a stable environment of 22(±2)° C., 60 (±10) % humidity and a 12:12 light:dark cycle; with lightson at 0600. Mice were housed in groups of four in standard cages with adlibitum access to food (Teklad: LM 485; Madison, Wis.) and water exceptas noted, for at least two weeks before the experiments.

All experiments were conducted between the hours of 0700 and 0900. Themice were food deprived (food removed at 1600 hr from all animals on dayprior to experiment) and thereafter individually housed. All micereceived an intraperitoneal injection (5 μl/kg) of either saline or testcompound at doses of 0.1, 1.0, 10, and 100 μg/kg, and were immediatelypresented with a pre-weighed food pellet (Teklad LM 485). The foodpellet was weighed at 30-minute, 1-hr, 2-hr and 6-hr intervals todetermine the amount of food eaten. The ED₅₀ for inhibition of foodintake over 30 min was determined for several test compounds, and theresults appear in Table II, below.

TABLE II GLP-1 Appetite Cyclase Suppression EC50 nM ED50 ug/kg exendin40.27 0.21 218 >1000 1.80 219 1.11 0.08 220 0.8 0.12 221 0.69 6.70 2222.70 weak 223 0.46 2.40 224 3.22 weak 225 23 weak 226 102 2.40 227 149NA 228 458 NA 229 60.4 14.50  230 157 NA

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art which are encompassed within the spirit ofthe invention are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference in its entirety to the same extent as if each individualpublication was specifically and individually indicated to be soincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims.

We claim: 1-35. (canceled)
 36. A compound comprising exendin-4, oragonist analog of exendin-4, linked to a polyamino acid through theC-terminal amino acid of the exendin-4 or agonist analog of exendin-4,wherein the agonist analog of exendin-4 comprises one or more naturallyoccurring amino acids eliminated or replaced with another amino acid.37. The compound according to claim 36, wherein the polyamino acid isselected from the group consisting of poly(L-lysine), poly-glutamicacid, and poly-aspartic acid.
 38. The compound according to claim 36,wherein the polyamino acid is poly(L-lysine).
 39. A pharmaceuticalcomposition comprising the exendin-4, or agonist analog of exendin-4according to claim 36 and a pharmaceutically acceptable carrier whereinthe exendin-4, or agonist analog of exendin-4 has a kidney clearancethat is less than 50% of the kidney clearance of the exendin-4, oragonist analog of exendin-4 without the C-terminal linked polyaminoacid.
 40. A method for treating diabetes, postprandial hyperglycemia, orimpaired glucose tolerance comprising administering to a human in needof treatment for diabetes, postprandial hyperglycemia, or impairedglucose tolerance a therapeutically effective amount of a compositionaccording to claim
 39. 41. The method for treating diabetes according toclaim
 40. 42. The method according to claim 41, wherein the diabetes istype II diabetes.
 43. A compound comprising an agonist analog ofexendin-4, wherein said agonist analog of exendin-4 comprises the aminoacid sequence of SEQ ID NO.: 200, wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Ala Xaa₁₉Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ Xaa₂₆ Xaa₂₇ Xaa₂₈-Z1; wherein: Xaa₁is His, Arg, Tyr, Ala, Norval, Val or Norleu; Xaa₂ is Ser, Gly, Ala orThr; Xaa₃ is Ala, Asp or Glu; Xaa₄ is Ala, Norval, Val, Norleu or Gly;Xaa₅ is Ala or Thr; Xaa₆ is Phe, Tyr or naphthylalanine; Xaa₇ is Thr orSer; Xaa₈ is Ala, Ser or Thr; Xaa₉ is Ala, Norval, Val, Norleu, Asp orGlu; Xaa₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met; Xaa₁₁ is Ala orSer; Xaa₁₂ is Ala or Lys; Xaa₁₃ is Ala or Gln; Xaa₁₄ is Ala, Leu, Ile,pentylglycine, Val or Met; Xaa₁₅ is Ala or Glu; Xaa₁₆ is Ala or Glu;Xaa₁₇, is Ala or Glu; Xaa₁₉ is Ala or Val; Xaa₂₀ is Ala or Arg; Xaa₂₁ isAla or Leu; Xaa₂₂ is Phe, Tyr or naphthylalanine; Xaa₂₃ is Ile, Val,Leu, pentylglycine, tert-butylglycine or Met; Xaa₂₄ is Ala, Glu or Asp;Xaa₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine; Xaa₂₆ is Ala or Leu;Xaa₂₇ is Ala or Lys; Xaa₂₈ is Ala or Asn; Z₁ is —OH, —NH₂, Gly Gly-Z₂,Gly Gly Xaa₃₁-Z₂, Gly Gly Xaa₃₁ Ser-Z₂, Gly Gly Xaa₃₁ Ser Ser-Z₂, GlyGly Xaa₃₁ Ser Ser Gly-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala-Z₂, Gly GlyXaa₃₁ Ser Ser Gly Ala Xaa₃₆-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆Xaa₃₇-Z₂, Gly Gly Xaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈-Z₂ or Gly GlyXaa₃₁ Ser Ser Gly Ala Xaa₃₆ Xaa₃₇ Xaa₃₈ Xaa₃₉-Z₂ [SEQ ID NO.: 203]wherein: Xaa₃₁, Xaa₃₆, Xaa₃₇ and Xaa₃₈ are independently Pro,homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,N-alkylpentylglycine or N-alkylalanine; and; Z₂ is —OH or —NH₂; providedthat no more than three of Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₈, Xaa₉, Xaa₁₀,Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₁₉, Xaa₂₀, Xaa₂₁,Xaa₂₄, Xaa₂₅, Xaa₂₆, Xaa₂₇ and Xaa₂₈ are Ala; and provided also that, ifXaa₁ is His, Arg or Tyr, then at least one of Xaa₃, Xaa₄ and Xaa₉ isAla; and Xaa₃₉ is Ser or Tyr.
 44. The compound according to claim 43wherein the agonist analog of exendin-4 is linked to a polyamino acidthrough the C-terminal amino acid of said agonist analog of exendin-4.45. The compound according to claim 44, wherein the polyamino acid isselected from the group consisting of poly(L-lysine), polyglutamic acid,and poly-aspartic acid.
 46. The compound according to claim 45, whereinthe polyamino acid is poly(L-lysine).
 47. A pharmaceutical compositioncomprising the agonist analog of exendin-4 according to claim 43 and apharmaceutically acceptable carrier wherein the agonist analog ofexendin-4 has a kidney clearance that is less than 50% of the kidneyclearance of the agonist analog of exendin-4 without the C-terminallinked polyamino acid.
 48. A method for treating diabetes, postprandialhyperglycemia, or impaired glucose tolerance comprising administering toa human in need of treatment for diabetes, postprandial hyperglycemia,or impaired glucose tolerance a therapeutically effective amount of acomposition according to claim
 47. 49. The method for treating diabetesaccording to claim
 48. 50. The method of claim 49, wherein the diabetesis type II diabetes.